JP5501954B2 - Δ9 elongase and their use in making polyunsaturated fatty acids - Google Patents

Description

CROSS REFERENCE TO RELATED APPLICATIONS This specification incorporates the priority of US Provisional Patent Application No. 60 / 911,925, filed Apr. 16, 2007, the entire contents of which are incorporated herein by reference. Insist.

  The present invention is in the field of biotechnology. More specifically, the present invention relates to the identification of polynucleotide sequences encoding Δ9 fatty acid elongases and the use of these elongases in the production of long chain polyunsaturated fatty acids (PUFA).

  Today, a variety of different hosts are being investigated as a means of commercial PUFA production, including plants, algae, fungi, stramenopile, and yeast. Genetic engineering is limited to the natural capacity of some hosts (naturally linoleic acid (LA; 18: 2 ω-6) or α-linolenic acid (ALA; 18: 3 ω-3) fatty acid production. Even) can be substantially modified to provide high level production of various long chain ω-3 / ω-6 PUFAs. Regardless of whether this is a natural ability or the result of recombinant technology, arachidonic acid (ARA; 20: 4 ω-6), eicosapentaenoic acid (EPA; 20: 5 ω-3), and docosahexaenoic acid ( Production of DHA; 22: 6 ω-3) may require expression of Δ9 elongase.

Most Δ9 elongase enzymes identified to date have both converted LA to eicosadienoic acid (EDA; 20: 2 ω-6) and ALA to eicosatrienoic acid (ETrA; 20: 3 ω-3). Competent (following reaction with Δ8 desaturase, dihomo-γ-linolenic acid (DGLA; 20: 3 ω-6) and eicosatetraenoic acid (ETA; 20: 4 ω-3) from EDA and ETrA Each subsequently synthesized, and following reaction with Δ5 desaturase, ARA and EPA are subsequently synthesized from DGLA and ETA, respectively, and DHA synthesis requires subsequent expression of additional _{C20 / 22} elongase and Δ4 desaturase) .

  Despite the need for new methods of generating ARA, EPA, and DHA, only a few Δ9 elongase enzymes have been identified. The Δ9 elongase from Isochrysis galvana is publicly available (described in Genbank accession number AAL37626 and Patent Documents 1, 2, 3, and 4). U.S. Patent Nos. 5,099,028 and 6,057, 196, co-pending applications of assignees of the Applicants, disclose a Δ9 elongase from Euglena gracilis and Eureptiella sp. CCMP389, and a Δ9 elongase motif.

International Publication No. 02/077213 Pamphlet International Publication No. 2005/083093 Pamphlet International Publication No. 2005/012316 Pamphlet International Publication No. 2004/057001 Pamphlet International Publication No. 2007/061845 Pamphlet International Publication No. 2007/061742 Pamphlet

  Thus, there is a need to identify and isolate additional genes encoding Δ9 elongases suitable for heterologous expression in a variety of host organisms for use in the production of ω-3 / ω-6 fatty acids.

  Applicants have solved the stated problem by isolating a gene encoding a Δ9 fatty acid elongase from Euglena anabena.

The present invention relates to new genetic constructs encoding polypeptides having Δ9 elongase activity, and their use in algae, bacteria, yeast, Euglena, stramenopile, and fungi to produce PUFAs. Therefore, the present invention
(A) a nucleotide having a Δ9 elongase activity and encoding a polypeptide having at least 80% amino acid identity based on the Clustal V method of alignment when compared to the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 Array,
(B) encodes a polypeptide having Δ9 elongase activity and has at least 80% sequence identity when compared to the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 26, based on the BLASTN method of alignment. A nucleotide sequence having,
(C) a nucleotide sequence encoding a polypeptide having Δ9 elongase activity and hybridizing under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 26, or (d) (a ), A microorganism comprising an isolated polynucleotide comprising the complement of the nucleotide sequence of (b) or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary A host cell is provided.

In another embodiment, the invention provides:
a) (i) a recombinant nucleotide that encodes a Δ9 elongase polypeptide and has at least 80% amino acid identity based on the Clustal V method of alignment when compared to the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 Providing a molecule, and (ii) a microbial host cell comprising a linoleic acid source;
b) growing the microbial host cell of step (a) under conditions in which a nucleic acid fragment encoding a Δ9 elongase polypeptide is expressed and linoleic acid is converted to eicosadienoic acid;
c) optionally providing a method of producing eicosadienoic acid comprising the step of recovering eicosadienoic acid of step (b).

In an additional embodiment, the present invention provides:
a) (i) a recombinant nucleotide that encodes a Δ9 elongase polypeptide and has at least 80% amino acid identity based on the Clustal V method of alignment when compared to the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 Providing a molecule, and (ii) a microbial host cell comprising an alpha-linolenic acid source;
b) growing the microbial host cell of step (a) under conditions in which a nucleic acid fragment encoding a Δ9 elongase polypeptide is expressed and α-linolenic acid is converted to eicosatrienoic acid;
c) optionally providing a method of producing eicosatrienoic acid comprising the step of recovering eicosatrienoic acid of step (b).

  In another embodiment, the invention encodes a Δ9 elongase set forth in SEQ ID NO: 26, wherein at least 98 codons are codon optimized for expression in a Yarrowia species. Provide molecules.

Representative omega-3 and omega-6 fatty acid biosynthetic pathways that lead to the conversion of myristic acid to DHA through various intermediates. Figure 2 shows a chromatogram of the lipid profile of Euglena anabena cell extract as described in the examples. A plasmid map of (A) pY115 (SEQ ID NO: 19), (B) pY159 (SEQ ID NO: 23), (C) pY173 (SEQ ID NO: 24) and (D) pY174 (SEQ ID NO: 25) is provided. A comparison of the nucleotide sequences of EaD9Elo (SEQ ID NO: 11) and EaD9ES (SEQ ID NO: 26) is shown. A comparison of the nucleotide sequences of EaD9Elo (SEQ ID NO: 11) and EaD9ES (SEQ ID NO: 26) is shown. The plasmid maps of (A) pEaD9ES (SEQ ID NO: 28) and (B) pZUFmEaD9eS (SEQ ID NO: 29) are provided.

  The present invention can be more fully understood from the following detailed description and the accompanying sequence descriptions, which form a part of this specification.

  The next sequence is 37C. F. R. 1. § 821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures—Sequence Rules”) and meet the World Intellectual Property Organization (WIPO) standard ST. 25 (1998), and the EPO and PCT sequence listing requirements (Rules 5.2 and 49.5 (a-2), and Detailed Regulations No. 208 and Annex C). The symbols and model used for nucleotide and amino acid sequence data are 37C. F. R. Follow the rules set forth in §1.822.

  SEQ ID NOs: 1-4, 9-19, and 22-29 are genes or proteins (or portions thereof) or plasmids that encode ORFs identified as shown in Table 1.

  SEQ ID NOs: 5 and 6 correspond to oligonucleotides oEugEL1-1 and oEugEL1-2, respectively, used for amplification of Euglena gracilis Δ9 elongase.

  SEQ ID NOs: 7 and 8 correspond to the M13F universal primer and primer M13-28Rev, respectively, used for terminal sequencing of Euglena anabena DNA inserts.

  SEQ ID NOs: 20 and 21 correspond to primers oYFBA1 and oYFBA1-6, respectively, used to amplify the FBAINm promoter from plasmid pY115.

  Disclosed herein are new Euglena anabena Δ9 elongase enzymes and genes encoding the same that may be used to manipulate biochemical pathways for the production of healthy PUFAs.

  PUFA or its derivatives are used as dietary substitutes, or nutritional supplements, especially infant formulas, for patients receiving intravenous nutrition or to prevent or treat malnutrition. Alternatively, purified PUFA (or a derivative thereof) may be incorporated into cooking oil, fat or margarine that is formulated to receive the desired amount of dietary supplements in normal use. PUFAs may also be incorporated into infant formula, nutritional supplements or other foods and may have use as anti-inflammatory or cholesterol-lowering agents. Optionally the composition may be used for pharmaceutical applications (human or veterinary).

Definitions In the context of this disclosure, a number of terms and abbreviations are used. The following definitions are provided:

  “Reading frame” is abbreviated ORF.

  “Polymerase chain reaction” is abbreviated PCR.

  “American Microbial Conservation Agency” is abbreviated ATCC.

  “Polyunsaturated fatty acid” is abbreviated PUFA.

  “Triacylglycerol” is abbreviated TAG.

  The terms "invention" or "invention" are not intended to limit the use of any particular embodiment of the invention to any one of the specific embodiments of the invention as used herein, and as stated in the claims and specification. In general, it applies to any and all embodiments of the invention.

  As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless indicated otherwise. Thus, for example, reference to “a plant” includes a plurality of such plants, reference to “a cell” includes one or more cells and equivalents known to those of skill in the art, and the like including.

The term “fatty acid” refers to long chain aliphatic acids (alkanoic acids) of various chain lengths of about C _{12 to} C ₂₂ (although both longer and shorter chain length acids are known). . The predominant chain lengths are between _C 16 _{-C 22.} “Saturated fatty acids” vs. “unsaturated fatty acids”, “monounsaturated fatty acids” vs. “polyunsaturated fatty acids” (or “PUFA”), and “omega-6 fatty acids” (ω-6 or n-6) ” The difference between “omega-3 fatty acids” (ω-3 or n-3) vs. “omega-3” is more fully defined in US Pat. No. 7,238,482.

  Fatty acids are described here by a simple notation system “X: Y”, where X is the total number of carbon (C) atoms of a particular fatty acid and Y is the number of double bonds. The number following the fatty acid name indicates the position of the double bond from the carboxyl terminus of the fatty acid, and the affix “c” indicates the cis configuration of the double bond (eg, palmitic acid (16: 0), stearic acid (18: 0) ), Oleic acid (18: 1, 9c), petrothelic acid (18: 1, 6c), LA (18: 2, 9c, 12c), GLA (18: 3, 6c, 9c, 12c), and ALA (18 : 3, 9c, 12c, 15c)). Unless otherwise noted, 18: 1, 18: 2, and 18: 3 refer to oleic acid, LA, and ALA fatty acids, respectively. Unless otherwise noted, double bonds are considered in the cis configuration. For example, the double bond in 18: 2 (9,12) is considered to be in the cis configuration.

  The nomenclature used to describe PUFAs in this disclosure is shown in Table 2 below. In the column entitled “abbreviation”, an omega-reference system is used to determine the double bond closest to the omega carbon, counting from the carbon number, double bond number, and omega carbon (number 1 for this purpose). Suggest location. The rest of the table summarizes the common names of omega-3 and omega-6 fatty acids and their precursors, the remaining abbreviations used throughout this specification, and the chemical names of each compound.

  The terms “triacylglycerol”, “oil”, and “TAG” refer to neutral lipids composed of three fatty acyl residues that esterify with a glycerol molecule (and such terms are disclosed in this disclosure). Are used interchangeably throughout.) Such oils can contain long chain PUFAs, as well as shorter saturated and unsaturated fatty acids, and longer chain saturated fatty acids. Thus, “oil biosynthesis” generally refers to TAG synthesis in cells.

  “Percent (%) PUFA in total lipid and oil fraction” refers to the percentage of PUFA relative to the total fatty acids in these fractions. The terms “total lipid fraction” or “lipid fraction” both refer to the sum of total lipids (ie, neutral and polar) in an oleaginous organism, so phosphatidylcholine (PC) fraction, phosphatidylethanolamine (PE) The fraction and lipids located within the triacylglycerol (TAG or oil) fraction are included. However, the terms “lipid” and “oil” are used interchangeably throughout this specification.

  A metabolic or biosynthetic pathway is, in the biochemical sense, formed in a cell and catalyzed by an enzyme to form a metabolite that is used or stored by the cell or to initiate another metabolic pathway (flux generation step and Can be viewed as a series of chemical reactions that achieve either Many of these routes are complex and involve step-by-step modification of the starting material into a product with the desired exact chemical structure.

The term "PUFA biosynthetic pathway" refers to oleic acid and omega-6 fatty acids such as LA, EDA, GLA, DGLA, ARA, DRA, DTA, and DPAn-6, and ALA, STA, ETrA, ETA, EPA, DPA. , And metabolic processes that convert to omega-3 fatty acids such as DHA. This process is described in detail in the literature (see e.g. WO 2006/052870). Briefly, this process involves carbon chain extension through the addition of carbon atoms by a series of special desaturases and extension enzymes (ie, “PUFA biosynthetic pathway enzymes”) present in the endoplasmic reticulum membrane, and Accompanied by molecular unsaturation through double bond addition. More specifically as "PUFA biosynthetic pathway enzyme", [Delta] 9 _{elongase, C 14/16 elongase, C 16/18 elongase, C 18/20 elongase, C 20/22} elongase, [Delta] 4 desaturase, .DELTA.5 desaturase, [Delta] 6 desaturase , Δ12 desaturase, Δ15 desaturase, Δ17 desaturase, Δ9 desaturase and / or Δ8 desaturase, and any of the enzymes associated with PUFA biosynthesis (and the gene encoding said enzyme).

  The term “ω-3 / ω-6 fatty acid biosynthetic pathway” refers to a set of genes that, when expressed under appropriate conditions, encode enzymes that catalyze the production of one or both of ω-3 and ω-6 fatty acids. Point to. A gene typically involved in the ω-3 / ω-6 fatty acid biosynthetic pathway encodes a PUFA biosynthetic pathway enzyme. A representative pathway is shown in FIG. 1, providing conversion of myristic acid to DHA via various intermediates, and how both omega-3 and omega-6 fatty acids can be generated from a common source To demonstrate. The pathway naturally divides into two parts, one part generates omega-3 fatty acids and the other part generates omega-6 fatty acids.

  The term “functionality” is used herein in the context of the ω-3 / ω-6 fatty acid biosynthetic pathway, where some (or all) of the genes in the pathway express the active enzyme and are biocatalyzed or substrates. Means to bring conversion. Since some fatty acid products only require the expression of a subset of genes in this pathway, the “ω-3 / ω-6 fatty acid biosynthetic pathway” or “functional ω-3 / ω-6 fatty acid biosynthetic pathway” Is understood not to imply that all PUFA biosynthetic pathway enzyme genes are required.

The term “Δ6 desaturase / Δ6 elongase pathway” includes at least one Δ6 desaturase and at least one C _18/20 elongase (also referred to as a Δ6 elongase), whereby GLA and / or STAs are intermediate fatty acids. As referring to the PUFA biosynthetic pathway that allows biosynthesis of DGLA and / or ETA from LA and ALA, respectively. With the expression of other desaturases and elongases, ARA, EPA, DPA, and DHA may also be synthesized.

  The term “Δ9 elongase / Δ8 desaturase pathway” includes at least one Δ9 elongase and at least one Δ8 desaturase, whereby EDA and / or ETrA as intermediate fatty acids, respectively, from LA and ALA to DGLA and / or It refers to the PUFA biosynthetic pathway that allows biosynthesis of ETA. With the expression of other desaturases and elongases, ARA, EPA, DPA, and DHA may also be synthesized.

  The term “intermediate fatty acid” refers to any fatty acid produced within this pathway that can be further converted into the intended product fatty acid by the action of other metabolic pathway enzymes in the fatty acid metabolic pathway. For example, when EPA is produced using the Δ9 elongase / Δ8 desaturase pathway, EDA, ETrA, DGLA, ETA, and ARA are produced, and these fatty acids can be further converted to EPA through the action of other metabolic pathway enzymes. So it can be regarded as an “intermediate fatty acid”.

  The term “byproduct fatty acid” refers to any fatty acid that is produced within a fatty acid metabolic pathway that is not the intended fatty acid product of the pathway and is not an “intermediate fatty acid” of the pathway. For example, when EPA is produced using the Δ9 elongase / Δ8 desaturase pathway, siadonic acid (SCI) and juniperonic acid (JUP) can also be produced by the action of Δ5 desaturase on either EDA or ETrA, respectively. They are considered “by-product fatty acids” because neither is further converted to EPA by the action of other metabolic pathway enzymes.

  The term “desaturase” refers to a polypeptide that can be desaturated, ie, introduce a double bond into one or more fatty acids, resulting in a fatty acid or precursor of interest. To refer to a specific fatty acid, it is more convenient to show the activity of desaturase by counting from the carboxyl terminus of the substrate using the delta system, despite the use of the ω reference system throughout this specification. The desaturase of interest can, for example, (1) desaturate fatty acids between the 8th and 9th carbon atoms, counting from the carboxyl terminus of the molecule, which can catalyze the conversion of EDA to DGLA and / or ETrA to ETA. (8) Δ5 desaturase that catalyzes the conversion of DGLA to ARA and / or ETA to EPA, (3) Δ6 desaturase that catalyzes the conversion of LA to GLA and / or ALA to STA, (4) DPA Δ4 desaturase that catalyzes the conversion of DTA to DHA and / or DTA to DPAn-6, (5) Δ12 desaturase that catalyzes the conversion of oleic acid to LA, (6) the conversion of LA to ALA and / or GLA to STA (15) from ARA to EPA and A Δ17 desaturase that catalyzes the conversion of DGLA to ETA, and / or a Δ9 desaturase that catalyzes the conversion of palmitic acid to palmitoleic acid (16: 1) and / or stearic acid to oleic acid (18: 1). Can be mentioned. In the art, Δ15 and Δ17 desaturases are also based on their ability to convert ω-6 fatty acids to their ω-3 counterparts (eg, LA to ALA and ARA to EPA, respectively) Sometimes also referred to as “omega-3 desaturase”, “w-3 desaturase” and / or “ω-3 desaturase”. In some embodiments, it is most desirable to empirically determine the specificity of a particular fatty acid desaturase by transforming an appropriate host with the fatty acid desaturase gene and determining its effect on the fatty acid profile of the host. It may be.

For purposes herein, enzymes that catalyze the first condensation reaction (ie, conversion of malonyl-CoA and long chain acyl-CoA to β-ketoacyl-CoA) are collectively referred to as “elongase”. In general, the substrate selectivity of elongases is somewhat broad, but is distinguished by both chain length and degree of unsaturation. Thus, elongases can have different specificities. For example, C _14/16 elongase _utilizes a C ₁₄ substrate (eg, myristic acid), C _16/18 elongase utilizes a C ₁₆ substrate (eg, palmitic acid), and C _18/20 elongase (a synonymous term) also known) utilizes _{C 18} substrate (e.g. GLA, STA) as Δ6 _{elongase, C 20/22} elongase utilizes a _{C 20} substrate (e.g. ARA, EPA). Similarly, of particular interest here, the “Δ9 elongase” catalyzes the conversion of LA to EDA and / or ALA to ETrA. It is important to note that some elongases have broad specificity and therefore a single enzyme may be able to catalyze several elongase reactions. Thus, for example, Δ9 elongase _also an alternative to _{C 16/18 elongase, C 18/20} elongase and / or Shirezu may function as _{C 20/22} elongase, although not preferred, .DELTA.5 and Δ6 fatty acids such as EPA and / or GLA May have each specificity. In a preferred embodiment, it may be desirable to empirically determine the specificity of a fatty acid elongase by transforming a suitable host with a fatty acid elongase gene and determining its effect on the fatty acid profile of the host. The elongase system generally comprises four enzymes that play the role of extending the fatty acid carbon chain to produce a fatty acid that is two carbons longer than the fatty acid substrate on which the elongase system acts. More specifically, the extension process occurs in the context of fatty acid synthase where CoA is an acyl carrier (Lassner et al., Plant Cell, 8: 281-292 (1996)). In the first step, which is known to be substrate specific and rate limiting, malonyl-CoA is condensed with long chain acyl-CoA to extend carbon dioxide (CO ₂ ) and (acyl moiety is extended by 2 carbon atoms). Resulting in β-ketoacyl-CoA. Subsequent reactions include a reduction to β-hydroxyacyl-CoA, a dehydration to enoyl-CoA, and a second reduction resulting in prolonged acyl-CoA. Examples of reactions catalyzed by the elongase system are the conversion of GLA to DGLA, STA to ETA, LA to EDA, ALA to ETrA, ARA to DTA, and EPA to DPA.

  For purposes herein, the term “EaD9Elo1” refers to the Δ9 elongase enzyme (SEQ ID NO: 13) isolated from Euglena anabena and encoded by SEQ ID NO: 11 herein. The term “EaD9Elo2” refers to E.D. Refers to the Δ9 elongase enzyme (SEQ ID NO: 14) isolated from anabena and encoded by SEQ ID NO: 12 herein. Similarly, the term “EaD9eS” refers to E.D. Refers to a synthetic Δ9 elongase derived from anabena and codon-optimized for expression in Yarrowia lipolytica (ie, SEQ ID NOs: 26 and 27).

The terms “conversion efficiency” and “% substrate conversion” refer to the efficiency with which a particular enzyme (eg, elongase) can convert a substrate into a product. The conversion efficiency is measured according to the following formula: ([Product] / [Substrate + Product]) ^* 100, where “product” includes the immediate product and all products in the pathway derived therefrom.

  The term “oily” refers to organisms that tend to conserve their energy sources in the form of lipids (Weete, “Fungal Lipid Biochemistry”, 2nd edition, Plenum, 1980). Within oleaginous microorganisms, cellular oil or TAG content generally follows a sigmoidal curve, and lipid concentration increases until it reaches a maximum in late logarithmic or early stationary growth phase, and then gradually decreases in late quiescent and dead phases (Yongmanithai and Ward, Appl. Environ. Microbiol., 57: 419-25 (1991)). It is not uncommon for oleaginous microorganisms to accumulate more than about 25% of their dry cell weight as oil.

  The term “oleaginous yeast” refers to microorganisms classified as yeast that make oil. Examples of oleaginous yeasts include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lichospose. It is not limited to this in any way.

  The term “Euglenophyceae” refers to a group of unicellular colorless or photosynthetic flagellates (“Euglena”) that inhabit freshwater, ocean, soil, and parasitic environments. The class is characterized by single living cells, mostly free-swimming, with two flagellums that grow from an anterior indentation known as a reservoir, one of which may be non-emergent. . The photosynthetic Euglena genus contains one to many chloroplasts, which vary from microdiscs to a wide range of plates or ribbons. The colorless Euglena genus relies on osmotic or feeding nutrition for nutrient assimilation. About 1000 species have been described and classified into about 40 genera and 6 eyes. Examples of Euglenophyceae include, but are not limited to, the genus Euglena, Eutreptilla, and Tetrepteptia.

  As used herein, “nucleic acid” refers to a polynucleotide and includes single- or double-stranded polymers of deoxyribonucleotides or ribonucleotide bases. The nucleic acid may also include fragments and modified nucleotides. Thus, the terms “polynucleotide”, “nucleic acid sequence”, “nucleotide sequence” or “nucleic acid fragment” are used interchangeably and are single-stranded or double-stranded, optionally containing synthetic, non-natural or modified nucleotide bases. Refers to a polymer of RNA or DNA contained. A polynucleotide in the form of a DNA polymer may comprise one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (usually found in their 5 'monophosphate form) are referred to by their single letter names as follows: “A” is adenylate or deoxyadenylate (for RNA or DNA respectively), “C” is cytidylate or deoxycytidylate, “G” is guanylate or deoxyguanylate, “U” is uridylate, “T” "Deoxythymidylic acid", "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for Inosine, and “N” is any nucleotide.

  The term “conserved domain” or “motif” refers to a set of amino acids that are conserved at a particular position along an aligned sequence of evolutionarily related proteins. While amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions suggest amino acids that are essential in the structure, stability, or activity of the protein. Because they are identified by their high degree of conservation in matched sequences of the protein homolog family, using them as identifiers or “signatures”, proteins of newly determined sequences were previously identified Whether it belongs to the protein family can be determined. WO 2007/061845 and WO 2007/061742 describe characteristic motifs associated with Δ9 elongases.

  The terms “homology”, “homologous”, “substantially similar”, and “substantially identical” are used interchangeably herein. They refer to nucleic acid fragments in which changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a particular phenotype. These terms also alter the nucleic acid fragment of the invention, such as deletion or insertion of one or more nucleotides, that does not substantially change the functional properties of the resulting nucleic acid fragment compared to the original native fragment. Also refers to. Thus, as will be appreciated by those skilled in the art, the present invention encompasses more than specific exemplary sequences.

  In addition, one of ordinary skill in the art will appreciate that the substantially similar nucleic acid sequences encompassed by the present invention are nucleotide equivalents disclosed herein that are functionally equivalent to either the sequences exemplified herein or the nucleic acid sequences disclosed herein. Recognize that they are also defined by their ability to hybridize to any part of the sequence (eg, under moderately stringent conditions such as 0.5 × SSC, 0.1% SDS, 60 ° C.). . Adjust stringency conditions to screen moderately similar fragments, such as homologous sequences from distantly related organisms, or highly similar fragments, such as genes that replicate functional enzymes from closely related organisms . Post-hybridization washes determine stringency conditions.

  The term “selectively hybridize” is a definition of a nucleic acid sequence under stringent hybridization conditions that is detectably greater than its hybridization to a non-target nucleic acid sequence (eg, at least twice background). To the nucleic acid target sequence, and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have at least about 80% sequence identity with each other, or 90% sequence identity, and up to 100% sequence identity (ie, completely complementary).

  The term “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which the probe selectively hybridizes to its target sequence. Stringent conditions are sequence-dependent and will be different in different circumstances. By preparing hybridization stringency and / or wash conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatches in the sequence so that a lower degree of similarity is detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, and optionally less than 500 nucleotides in length.

  Typically stringent conditions include a pH of 7.0 to 8.3 in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or The temperature is at least about 30 ° C. for short probes (eg, 10-50 nucleotides) and at least about 60 ° C. for long probes (eg, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30-35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C., and 1 × -2 × SSC at 50-55 ° C. Washing in (20 × SSC = 3.0 M NaCl / 0.3 M trisodium citrate) is mentioned. Exemplary moderate stringency conditions include 40-45% formamide at 37 ° C., hybridization in 1M NaCl, 1% SDS, and washing with 0.5 × -1 × SSC at 55-60 ° C. Can be mentioned. Exemplary high stringency conditions include 50% formamide at 37 ° C., hybridization in 1M NaCl, 1% SDS, and washing with 0.1 × SSC at 60-65 ° C.

Specificity is typically a function of post-hybridization washes, with important factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, _Tm is determined by Meinkoth et al., Anal. Biochem. 138: 267-284 (1984), T _m = 81.5 ° C. + 16.6 (log M) +0.41 (% GC) −0.61 (% form) −500 / L (where M is (Molar concentration of monovalent cation,% GC is the percentage of guanosine and cytosine nucleotides in DNA,% form is the percentage of formamide in the hybridization solution, and L is the hybrid length in base pairs) Can be approximated by T _m is the temperature at which 50% of the complementary target sequence (under a defined ionic strength and pH) hybridizes with a perfectly matched probe. The T _m is reduced by about 1 ℃ per 1% mismatch, respectively. Thus, T _m , hybridization and / or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if a sequence with ≧ 90% identity is desired, the T _m can be reduced by 10 ° C. Generally, stringent conditions are selected to be about 5 ° C. lower than the thermal melting point (T _m ) for the specific sequence and its complement at a defined ionic strength and pH. However, stringent conditions can utilize hybridization and / or washing at 1, 2, 3, or 4 ° C. below the thermal melting point (T _m ), while moderately stringent conditions can be achieved using the thermal melting point (T _m). 6), 7, 8, 9, or 10 ° C less hybridization and / or washing than is possible, and low stringency conditions are 11, 12, 13, 14, 15, or 20 below the thermal melting point (T _m ) Low hybridization and / or washing can be used. Using the formula, hybridization and washing composition, and desired T _m , one of ordinary skill in the art will understand that variations in stringency of hybridization and / or washing solutions are essentially stated. Let's go. If the desired degree of mismatching results in a T _m of less than 45 ° C. (aqueous solution) or 32 ° C. (formamide solution), it is preferable to increase the SSC concentration so that higher temperatures can be used. Nucleic acid hybridization of detailed guide, Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York ( 1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Greene Publishing and Wiley-Intersc. ence, located in New York (1995 years). Hybridization and / or wash conditions can be applied for at least 10, 30, 60, 90, 120 or 240 minutes.

  “Sequence identity” or “identity” in the context of nucleic acid or polypeptide sequences refers to nucleobases or amino acid residues within two sequences that are identical when aligned for maximum correspondence over a defined comparison window. .

  Thus, the “sequence identity” percentage refers to a value determined by comparing two optimally matched sequences on a comparison window, where a portion of a polynucleotide or polypeptide sequence within the comparison window is two sequences May include additions or deletions (ie, gaps) as compared to a reference sequence (not including additions or deletions) for optimal alignment. Percentage is determined by determining the number of matching positions by determining the number of positions where the same nucleobase or amino acid residue is present in both sequences, and dividing the number of matching positions in the comparison window by the total number of positions. The result is calculated by multiplying the result by 100. Useful examples of percent sequence identity are 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any integer percentage between 50% and 100% However, it is not limited to this. These identities can be determined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations are described in DNASTAR Inc. Using various comparison methods designed to detect homologous sequences, including, but not limited to, the MEGAlign ^™ program of the LASERGENE bioinformatics suite from (Madison, WI) May be determined. In the context of this specification, when sequence analysis software is used for analysis, it is understood that the analysis results are based on the “default values” of the referenced program, unless otherwise noted. As used herein, “default value” means any value or set of parameters that is initially loaded with the software when it is first initialized.

“Alignment Clustal V method” is an alignment method labeled as Clustal V (Higgins and Sharp, CABIOS, 5: 151-153 (1989); Higgins, DG et al., Compute. Appl. Biosc., 8 189-191 (1992)), and in the MEGAlign ^™ program of the LASERGENE bioinformatics computation suite (supra). For multiple alignment, the default values correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The default parameters for pairwise alignment of protein sequences and calculation of% identity using the Clustal V method are KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5, and DIAGONALS SAVED = 5. For nucleic acids, these parameters are KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4, and DIAGONALS SAVED = 4. After sequence alignment using the Clustal V program, “% identity” can be obtained by looking at the “Sequence Distance” table in the same program.

  The “BLASTN method of alignment” is an algorithm for comparing nucleotide sequences using default parameters provided by the National Center for Biotechnology Information (NCBI).

  One skilled in the art will appreciate that many levels of sequence identity are useful in identifying polypeptides from other species that have the same or similar function or activity. Examples of useful% identity include 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer between 50% and 100% A percentage may be mentioned, but the present invention is not limited to this. In fact, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, Any integer amino acid identity between 50% and 100% may be useful in describing the present invention. Also of interest is any full-length or partial complement of this isolated nucleotide fragment.

  “Codon degeneracy” refers to the property of the genetic code that allows for variations in nucleotide sequence that do not affect the amino acid sequence of the encoded polypeptide. Accordingly, the present invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the Euglena polypeptide set forth in SEQ ID NO: 13 and SEQ ID NO: 14. Those skilled in the art are familiar with the “codon bias” exhibited by a particular host cell in the use of nucleotide codons to specify a particular amino acid. Therefore, when a gene is synthesized for improved expression in a host cell, it is desirable to design the gene so that its codon usage is close to the preferred codon usage of the host cell.

  “Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments, which are then assembled enzymatically to construct the entire gene. Thus, genes can be individually tailored for optimal gene expression based on nucleotide sequence optimization to reflect the codon bias of the host cell. One skilled in the art understands the potential for successful gene expression when the codon usage is biased to the codon preferred by the host. Preferred codon determination can be based on a search for genes from host cells for which sequence information is available.

  “Gene” refers to a nucleic acid fragment that expresses a particular protein, which may refer only to the coding region, or precedes the coding sequence (5 ′ non-coding sequence) and subsequent (3 ′ non-coding sequence) control. An array may be included. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Thus, a chimeric gene may comprise regulatory and coding sequences from different sources or from the same source but arranged in a manner different from that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene that is not normally found in the host organism, but has been introduced into the host organism by gene transfer. The foreign gene can comprise a natural gene inserted into a non-natural organism, or a chimeric gene. A “transgene” is a gene that has been introduced into the genome by transformation. A “codon optimized gene” is a gene whose codon usage is designed to mimic the preferred codon usage of the host cell.

  “Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. A “control sequence” is located upstream (5 ′ non-coding sequence), within or downstream (3 ′ non-coding sequence) of a coding sequence and transcription, RNA processing or stability, or translation of the associated coding sequence. Refers to a nucleotide sequence that affects Control sequences include, but are not limited to, promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem loop structures.

  “Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3 'to a promoter sequence. A promoter may be derived from a natural gene in its entirety, may be composed of different elements from different promoters found in nature, or may comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may direct gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. Promoters that cause gene expression at almost all developmental stages are commonly referred to as “structural promoters”. In most cases, it is further recognized that several variations of DNA fragments may have the same promoter activity, since the exact boundaries of the control sequences, in particular the 5 'end, are not fully defined.

  A promoter sequence may consist of proximal and more distal upstream elements, the latter elements often referred to as enhancers and / or silencers. Thus, an “enhancer” is a DNA sequence that can stimulate promoter activity and can be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or step-specific activity of the promoter. A “silencer” is a DNA sequence that can suppress promoter activity and may be an innate element of the promoter or a heterologous element inserted to suppress promoter level or step-specific activity.

  A “translation leader sequence” refers to a polynucleotide sequence located between the transcription start site of a gene and a coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation initiation sequence. Translation leader sequences may affect the processing of primary transcripts to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, GD, Mol. Biotechnol., 3: 225-236 (1995)).

  The terms “3 ′ untranslated sequence”, “termination sequence” and “transcription terminator” refer to a DNA sequence located downstream of the coding sequence. This includes polyadenylation recognition sequences that encode regulatory signals that can affect mRNA processing or gene expression, and other sequences. Polyadenylation signals are usually characterized by affecting the addition of polyadenylate tracts to the 3 'end of the mRNA precursor. The 3 'region can affect transcription, RNA processing or stability, or translation of the associated coding sequence.

  “RNA transcript” refers to the product resulting from DNA sequence transcription catalyzed by RNA polymerase. When an “RNA transcript” is a complete complementary copy of a DNA sequence, it is referred to as a primary transcript. If the RNA transcript is an RNA sequence derived from post-transcriptional processing of the primary transcript, it is referred to as mature RNA. “Messenger RNA” or “mRNA” refers to the RNA that is without introns and that can be translated into protein by the cell. “CDNA” refers to DNA that is complementary to and synthesized from mRNA template using reverse transcriptase. The cDNA can be single stranded or can be converted to double stranded form using the Klenow fragment of DNA polymerase I. “Sense” RNA refers to RNA transcript that includes the mRNA and can be translated into protein in the cell or in vitro. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (US Pat. No. 5,107,065). The complementarity of the antisense RNA may be complementarity to any part of a particular gene transcript, i.e. 5 'non-coding sequence, 3' non-coding sequence, intron, or coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but still affects cellular processes. The terms “complement” and “reverse complement” are used interchangeably herein with respect to mRNA transcripts and are intended to define the antisense RNA of the message.

  The term “operably linked” refers to a sequence of nucleic acid sequences on a single nucleic acid fragment such that one function is affected by the other function. For example, if a promoter can affect the expression of a coding sequence (ie, the coding sequence is under transcriptional control of the promoter), it is operably linked to that coding sequence. Coding sequences can be operably linked to control sequences in sense or antisense orientation.

  The term “recombinant” refers to an artificial combination of two otherwise separate sequence segments, eg, by chemical synthesis or by manipulation of an isolated segment of nucleic acid through genetic engineering techniques.

  The term “expression” as used herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression also refers to translation of mRNA into protein (either precursor or mature).

  “Mature” protein refers to a post-translationally processed polypeptide, ie, one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA, ie, with pre- or propeptides still present. The pre- or propeptide may be (but is not limited to) an intracellular localization signal.

  The terms “plasmid” and “vector” refer to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell, usually in the form of circular double-stranded DNA fragments. Such elements may be single or double stranded DNA or RNA linear or circular, self-replicating sequences, genomic integration sequences, phage or nucleotide sequences from any source, some of which Are combined or recombined into a unique structure that allows the expression cassette to be introduced into the cell.

  The term “expression cassette” refers to the coding sequence of the selected gene and the preceding (5 ′ non-coding sequence) and subsequent (3 ′ non-coding sequence) necessary for the expression of the selected gene product. ) Refers to a DNA fragment comprising regulatory sequences. Thus, an expression cassette typically consists of (1) a promoter sequence, (2) a coding sequence (ie ORF), and (3) a 3 ′ untranslated region (ie terminator) that usually contains a polyadenylation site in eukaryotes. Composed. An expression cassette is usually included in the vector to facilitate cloning and transformation. Different expression cassettes can be transformed into different organisms including bacteria, yeast, plants, and mammalian cells, as long as appropriate control sequences are used for each host.

  A “recombinant DNA construct” (also referred to herein as “expression construct” and “construct”) includes an artificial combination of nucleic acid fragments, such as regulatory and coding sequences that are not found together in nature, for example. Become. For example, a recombinant DNA construct may comprise control and coding sequences derived from different sources, or control and coding sequences derived from the same source but arranged in a manner different from that found in nature. Such a construct may be used alone or in conjunction with a vector. When using vectors, the choice of vector depends on the method used to transform the host cells, as is well known to those skilled in the art. For example, a plasmid vector can be used. Those skilled in the art will know the genetic elements that must be present on the vector in order to successfully transform, select, and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. I know enough. One skilled in the art will recognize that different independent transformation events will result in different expression levels and patterns (Jones et al., EMBOJ., 4: 2411-2418 (1985); De Almeida et al., Mol. Gen. Genetics, 218: 78-86 (1989)), and therefore it will also be recognized that multiple events must be screened in order to obtain a strain that exhibits the desired expression level and pattern. Such a screening may be achieved in particular by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblot analysis of protein expression, or phenotypic analysis.

  The term “introduced” means providing a cell with a nucleic acid (eg, an expression cassette) or protein. Introduced includes a reference to the integration of a nucleic acid into a eukaryotic or prokaryotic cell, where the nucleic acid may be integrated into the cell's genome and includes a reference to the temporary provision of the nucleic acid or protein into the cell. . Introduced includes stable or transient transformation methods, as well as references to sexual crossing. Thus, “introduced” in the context of insertion of a nucleic acid fragment (eg, a recombinant DNA construct or expression cassette) into a cell means “transfection” or “transformation” or “transduction”, eukaryotic or prokaryotic. Including reference to integration of the nucleic acid fragment into the cell, where the nucleic acid fragment is integrated into the genome of the cell (eg, chromosome, plasmid, plastid or mitochondrial DNA), converted to an autonomous replicon, or It may be transiently expressed (eg, transfected mRNA).

  As used herein, “gene transfer” refers to a cell comprising a heterologous polynucleotide in its genome. Preferably, the heterologous polynucleotide is stably integrated into the genome so that the polynucleotide is inherited by subsequent generations. Heterologous polynucleotides may be integrated into the genome alone or as part of an expression cassette. Gene transfer is modified by the presence of a heterologous nucleic acid, including a gene transfer so modified from the outset, as well as that produced from the first gene transfer by mating with a heterologous mating type. Any cell or cell line that is present is used herein. The term “gene transfer” as used herein is a genome (chromosome or extrachromosomal) due to a natural event such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant gene translocation, or spontaneous mutation. ) Is not included.

  Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in Sambrook, J. et al. , Fritsch, E .; F. And Maniatis, T .; Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratories: Cold Spring Harbor, NY (1989); J. et al. Bennan, M .; L. And Enquist, L .; W. , Experiments with Gene Fusions, Cold Spring Harbor Laboratories: Cold Spring Harbor, NY (1984); and Ausubel, F .; M.M. Et al, Current Protocols in Molecular Biology, Green Publishing Assoc. and Wiley-Interscience, Hoboken, NJ (1987). Transformation methods are well known to those skilled in the art and are described in An Overview: Microbiological Biosynthesis Of Fatty Acids And Triaclyglycols below.

  In general, lipid accumulation in oleaginous microorganisms is triggered by the overall carbon to nitrogen ratio present in the growth medium. This process leading to the novel synthesis of free palmitic acid (16: 0) in oleaginous microorganisms is described in detail in US Pat. No. 7,238,482. Palmitic acid is a precursor of longer-chain saturated and unsaturated fatty acid derivatives formed through the action of elongase and desaturase (FIG. 1).

  TAG (the main storage unit of fatty acids) is formed by a series of reactions involving: 1. 1.) Esterification of one molecule of acyl-CoA to glycerol-3-phosphate by acyltransferase yields lysophosphatidic acid. 2.) Esterification of the second acyl-CoA molecule by acyltransferase results in 1,2-diacylglycerol phosphate (commonly identified as phosphatidic acid). 3.) Removal of phosphate by phosphatidic acid phosphatase yields 1,2-diacylglycerol (DAG). ) Addition of a third fatty acid by the action of acyltransferase forms TAG. A wide range of fatty acids can be incorporated into the TAG, including saturated and unsaturated fatty acids and short and long chain fatty acids.

Biosynthesis of omega fatty acids The metabolic process by which oleic acid is converted to omega-3 / omega-6 fatty acids involves carbon chain extension through carbon atom addition and molecular desaturation through double bond addition. This requires a series of special desaturation and extension enzymes present in the endoplasmic reticulum membrane. However, as shown in FIG. 1 and described below, there are often multiple alternative pathways for the production of specific ω-3 / ω-6 fatty acids.

Specifically, all pathways require an initial conversion of oleic acid to LA, the first ω-6 fatty acid, by Δ12 desaturase. Next, using the “Δ9 elongase / Δ8 desaturase pathway” and LA as a substrate, long chain ω-6 fatty acids are formed as follows. (1) LA is converted to EDA by Δ9 elongase, (2) EDA is converted to DGLA by Δ8 desaturase, (3) DGLA is converted to ARA by Δ5 desaturase, (4) ARA is converted by C _20/22 elongase (5) DTA is converted to DPAn-6 by Δ4 desaturase. Alternatively, the “Δ9 elongase / Δ8 desaturase pathway” can use ALA as a substrate to produce long chain ω-3 fatty acids as follows: (1) LA is converted to ALA, which is the first omega-3 fatty acid by Δ15 desaturase, (2) ALA is converted to ETrA by Δ9 elongase, (3) ETrA is converted to ETA by Δ8 desaturase, (4 ETA is converted to EPA by Δ5 desaturase, (5) EPA is converted to DPA by C _20/22 elongase, and (6) DPA is converted to DHA by Δ4 desaturase. Optionally, omega-6 fatty acids may be converted to omega-3 fatty acids, for example, ALA is produced from LA by Δ15 desaturase activity, and ETA and EPA are produced from DGLA and ARA, respectively, by Δ17 desaturase activity.

Alternative pathways for biosynthesizing ω-3 / ω-6 fatty acids utilize the Δ6 desaturase and the C _18/20 elongase (ie, the “Δ6 desaturase / Δ6 elongase pathway”). More specifically, LA and ALA may be converted to GLA and STA, respectively, by Δ6 desaturase, and then a C _18/20 elongase converts GLA to DGLA and / or STA to ETA. The downstream PUFA is subsequently formed as described above.

  The particular functionality that needs to be introduced into a particular host organism for the production of omega-3 / omega-6 fatty acids is the host cell (and its natural PUFA profile and / or desaturase / elongase profile) It depends on the availability of the substrate, and the desired end product. Since the PUFA produced by the former pathway lacks GLA and / or STA, for example, in some embodiments, expression of the Δ9 elongase / Δ8 desaturase pathway may be preferred as opposed to expression of the Δ6 desaturase / Δ6 elongase pathway. Absent.

One skilled in the art will be able to identify a variety of candidate genes that encode each enzyme desired for omega-3 / ω-6 fatty acid biosynthesis. Useful desaturase and elongase sequences may be derived from any source, for example isolated from natural sources (bacteria, algae, fungi, plants, animals, etc.), produced by semi-synthetic pathways, or novel (de novo ) Synthesized. Although the particular source of desaturase and elongase genes introduced into the host is not critical, considerations for selecting specific polypeptides having desaturase or elongase activity include the following. 1. ) Substrate specificity of the polypeptide; 2.) whether the polypeptide or its constituents are rate-limiting enzymes; 3.) Whether a desaturase or elongase is essential for the desired PUFA synthesis; 4.) cofactors required by the polypeptide, and / or ) Whether the polypeptide has been modified after its production (eg, by a kinase or prenyl transferase). The expressed polypeptide preferably has parameters that are compatible with the biochemical environment of its location in the host cell (see US Pat. No. 7,238,482 for further details).

  In further embodiments, it may also be useful to consider the conversion efficiency of each particular desaturase and / or elongase. More specifically, since each enzyme rarely functions with 100% efficiency in converting a substrate to product, the final lipid profile of the unrefined oil produced in the host cell is typically desired. Of ω-3 / ω-6 fatty acids, as well as various PUFAs consisting of various upstream intermediate PUFAs. Therefore, in optimizing the biosynthesis of the desired fatty acid, the conversion efficiency of each enzyme is also a variable to consider.

With each of the above considerations in mind, appropriate desaturase and elongase activities (eg Δ6 desaturase, C 6) according to publicly available literature (eg GenBank), patent literature and experimental analysis of organisms capable of producing PUFAs. _18/20 elongase, .DELTA.5 desaturase, [Delta] 17 desaturase, [Delta] 15 desaturase, [Delta] 9 desaturase, Delta] 12 _{desaturase, C 14/16 elongase, C 16/18} elongase, [Delta] 9 elongase, [Delta] 8 desaturase, [Delta] 4 desaturase, and _{C 20/22} elongase) having Candidate genes can be identified. These genes will be suitable for introduction into a particular host organism in order to allow or enhance the organism's PUFA synthesis.

Sequence Identification of a New Δ9 Elongase In the present invention, the nucleotide sequence encoding the Δ9 elongase is isolated from Euglena anabena as summarized in Table 3 below.

Therefore, the present invention
(A) a nucleotide having a Δ9 elongase activity and encoding a polypeptide having at least 80% amino acid identity based on the Clustal V method of alignment when compared to the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 Array,
(B) encodes a polypeptide having Δ9 elongase activity and has at least 80% sequence identity when compared to the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 26, based on the BLASTN method of alignment. (C) an isolated polynucleotide comprising (c) a complement of the nucleotide sequence of (a) or (b), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary Relates to nucleotides.

  In yet another aspect, the invention encodes a polypeptide having Δ9 elongase activity and is based on the BLASTN method of alignment when compared to the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 26. It relates to an isolated polynucleotide comprising a nucleotide sequence having at least 90% sequence identity.

  More preferred amino acid fragments that are at least about 80% -90% identical are particularly suitable, and sequences that are at least about 90% -95% identical are most preferred. Similarly, preferred Δ9 elongases that encode nucleic acid sequences corresponding to this ORF are at least about 80% -90% identical, most preferably sequences that are at least about 90% -95% identical, encoding the active protein.

  In an alternative embodiment, the EaD9Elo1 and / or EaD9Elo2 sequences can be codon optimized for expression in a particular host organism. As is well known in the art, this further optimizes enzyme expression in alternative hosts, as the use of codons preferred by the host can substantially enhance expression of the foreign gene encoding the polypeptide. This is a useful tool. In general, the preferred codon for the host is to determine which codon is most frequently used by examining the frequency of codon usage in the protein (preferably expressed at the highest level) in the particular host species of interest. Can be determined. The codons preferred in the host species can then be used to synthesize, in whole or in part, for example, the coding sequence of the polypeptide of interest having elongase activity. All (or some) DNA can be synthesized to remove any destabilizing sequences or regions of secondary structure present in the transcribed mRNA. All (or some) of the DNA can be synthesized to modify the base composition to be more favorable in the desired host cell.

  In one embodiment of the invention, EaD9Elo1 (SEQ ID NO: 11) was codon optimized for expression in Yarrowia lipolytica. This is because Y.C. This was possible based on previous determinations of lipolytica codon usage profiles, identification of preferred codons, and determination of consensus sequences around the “ATG” start codon (US Pat. No. 7,238,482 and See U.S. Pat. No. 7,125,672). The resulting synthetic gene is referred to as EaD9ES (SEQ ID NO: 26). The protein sequence encoded by the codon optimized Δ9 elongase gene (ie, SEQ ID NO: 27) is identical to the wild type protein sequence (ie, SEQ ID NO: 13). Using similar technology, A synthetic Δ9 elongase derived from EaD9Elo2 (SEQ ID NO: 12) can be generated for expression in lipolytica.

  Those skilled in the art will use the teachings herein to select a variety of suitable, based on wild type EaD9Elo1 and / or EaD9Elo2 sequences, for optimal expression in alternative (ie, other than Yarrowia lipolytica) hosts. Other codon optimized Δ9 elongase proteins could be created. The present invention therefore relates to any codon optimized Δ9 elongase protein derived from the wild type nucleotide sequence EaD9Elo1 (SEQ ID NO: 11) or EaD9Elo2 (SEQ ID NO: 12). This includes the nucleotide sequence set forth in SEQ ID NO: 26, which encodes a synthetic Δ9 elongase protein (ie, EaD9eS) and is codon optimized for expression in Yarrowia lipolytica, It is not limited to this. In alternative embodiments, the portion of the codon encoding EaD9Elo1 and / or EaD9Elo2 is modified to include plants or plant parts, algae, bacteria, alternative yeasts, Euglena, stramenopile or fungi, including It may be desirable to enhance gene expression in a host organism, including but not limited to.

Identification and Isolation of Homologues The present elongase sequence (ie EaD9Elo1, EaD9Elo2, or EaD9eS) or any part thereof, the sequence in the same or another bacterial, algal, fungal, Euglena or plant species Analysis software may be used to search for Δ9 elongase homologues. In general, such computer software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

  Alternatively, either the present elongase sequence or part thereof may be used as a hybridization reagent for the identification of Δ9 elongase homologues. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. The probes of the present invention are typically single stranded nucleic acid sequences that are complementary to the nucleic acid sequence to be detected. A probe is “hybridizable” with the nucleic acid sequence to be detected. The probe length may vary between 5 bases and tens of thousands of bases, but a probe length of typically about 15 bases to about 30 bases is suitable. Only a portion of the probe molecule need be complementary to the nucleic acid sequence to be detected. Furthermore, the complementarity between the probe and the target sequence may not be perfect. Hybridization also occurs between molecules that are incompletely complementary, so that some of the specific bases in the hybridized region do not pair with the appropriate complementary bases.

  Hybridization methods are well defined. Typically, the probe and sample must be mixed under conditions that allow nucleic acid hybridization. This involves contacting the probe with the sample in the presence of an inorganic or organic salt under appropriate concentration and temperature conditions. The probe and sample nucleic acid must be in contact for a sufficiently long time so that any possible hybridization between the probe and sample nucleic acid occurs. The concentration of probe or target in the mixture determines the time required for hybridization to occur. The higher the probe or target concentration, the shorter the required hybridization incubation time. Optionally, chaotropic agents may be added (guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, and cesium trifluoroacetate). If desired, formamide can typically be added 30-30% (v / v) to the hybridization mixture.

  A variety of hybridization solutions can be used. They typically consist of about 20-60% by volume polar organic solvent, preferably 30% by volume. Typical hybridization solutions include about 30-50% v / v formamide, about 0.15-1 M sodium chloride (eg, sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6- About 0.05-0.1M buffer (such as 9) and about 0.05-0.2% detergent (such as sodium dodecyl sulfate), or 0.5-20 mM EDTA, Pharmacia ( Pharmacol Inc.), FICOLL (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal), and serum albumin are used. Typical hybridization solutions also include about 0.1-5 mg / mL unlabeled carrier nucleic acid, nucleic acid DNA fragments (such as calf thymus or salmon sperm DNA, or yeast RNA), and optionally about Also included is 0.5-2% weight / volume glycine. Volume exclusion agents including various polar water-soluble or aqueous swelling agents (such as polyethylene glycol), anionic polymers (such as polyacrylate or polymethyl acrylate), anionic sugar polymers (such as dextran sulfate) Other additives such as may be included.

  Nucleic acid hybridization can be adapted to a variety of assay formats. One of the most suitable is the sandwich assay format. Sandwich assays are particularly adaptable for hybridization under non-denaturing conditions. The main component of a sandwich type assay is a solid support. The solid support adsorbs or covalently binds an immobilized nucleic acid probe that is unlabeled and complementary to a portion of the sequence.

  In yet another embodiment, using any of the Δ9 elongase nucleic acid fragments described herein (or any homologues identified thereof), from the same or another bacterial, algal, fungal, Euglena or plant species, A gene encoding a homologous protein may be isolated. Isolation of homologous genes using sequence dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1. Nucleic acid hybridization method, 2. ) DNA and RNA amplification methods as exemplified in various uses of nucleic acid amplification techniques [eg, polymerase chain reaction of US Pat. No. 4,683,202 to Mullis et al.] (PCR); Tabor, S . Et al., Proc. Natl. Acad. Sci. U. S. A. , 82, 1074 (1985) ligase chain reaction (LCR); or (Walker et al., Proc. Natl. Acad. Sci. USA, 89, 392 (1992), chain displacement amplification (SDA). And 3.) Library construction and screening methods by complementation.

  For example, using any or all of the nucleic acid fragments as DNA hybridization probes to screen the library using methods well known to those skilled in the art, eg any desired yeast or fungi (EDA and (Or organisms that produce ETrA are preferred) and genes encoding proteins or polypeptides similar to the Δ9 elongase described herein can be isolated directly. Specific oligonucleotide probes based on this nucleic acid sequence can be designed and synthesized by methods known in the art (Maniatis, supra). Further, DNA probes can be synthesized directly using the entire sequence by methods known to those skilled in the art (eg, random primer DNA labeling, nick translation or end labeling techniques) or RNA probes can be synthesized using available in vitro transcription systems Can be synthesized. In addition, specific primers can be designed and used to amplify a portion (or full length) of this sequence. The resulting amplification product can be labeled directly during the amplification reaction or labeled after the amplification reaction and used as a probe under appropriate stringency conditions to isolate a full length DNA fragment.

  Typically in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the primer sequence should be designed to provide efficient and faithful replication of the target nucleic acid. PCR primer design methods are common and well known in the art. (From K. E. Davis, “Human Genetic Diseases: A Practical Approach”, Thein and Wallis, 1988). VA; and White, BA, edited by “Methods in Molecular Biology”, Rychlik, W., “PCR Protocols: Current Methods and Applications” (1993) Vol. 15, 31-39, Humana: To. ).

  In general, two short fragments of this sequence may be used in a PCR protocol to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. In addition, for the cloned nucleic acid fragment library, one primer sequence is derived from the nucleic acid fragment, and another primer sequence is a polyadenylate tract at the 3 ′ end of an mRNA precursor encoding a eukaryotic gene. PCR may be performed utilizing the presence of

  As an alternative, the second primer sequence may be based on a sequence derived from a cloning vector. For example, those skilled in the art will use PCR to detect one point and the 3 ′ or 5 ′ end of a transcript using PCR according to the RACE protocol (Frohman et al., Proc. Acad. Sci. USA, 85: 8998 (1988)). And a copy of the region between and can be amplified to create cDNA. Primers oriented in the 3 'and 5' directions can be designed from this sequence. Specific 3 'or 5' cDNA fragments can be isolated using 3'RACE or 5'RACE systems commercially available from Gibco / BRL (Gaithersburg, MD) (Ohara et al., Proc. Acad. Sci. U.S.). SA, 86: 5673 (1989); Loh et al., Science 243: 217 (1989)).

  In another embodiment, any Δ9 elongase nucleic acid fragment described herein (or any homologue thereof identified) may be used to create a new improved fatty acid elongase. As is well known in the art, mutations of the natural elongase gene can be obtained using in vitro mutagenesis and selection, chemical mutagenesis, “gene shuffling” methods or other means ( Such mutations include deletions, insertions, and point mutations, or combinations thereof). This results in the production of polypeptides with elongase activity that have more desirable physical and kinetic parameters, such as longer half-life or higher production rate of the desired PUFA, for function in host cells in vivo. Is possible. Or, if desired, through routine mutagenesis, expression of the resulting mutant polypeptides, and quantification of their activity, the region of the polypeptide of interest (ie, the Δ9 elongase) that is important for enzyme activity can be determined. An outline of these techniques is described in US Pat. No. 7,238,482. All such mutant proteins derived from EaD9Elo1, EaD9Elo2 and EaD9eS and the nucleotide sequences encoding them are within the scope of the present invention.

  Alternatively, an improved fatty acid may be synthesized by domain exchange, wherein a functional domain from any of the Δ9 elongase nucleic acid fragments described herein is replaced with a functional domain in the alternative elongase gene, thereby creating a new protein Is brought about. As used herein, a “domain” or “functional domain” refers to a nucleic acid sequence that can cause a biological response in a microorganism.

Methods for generating various omega-3 and / or omega-6 fatty acids Chimeric genes encoding the Δ9 elongase described herein (ie EaD9Elo1, EaD9Elo2, EaD9eS or other mutated enzymes, codon optimized, under appropriate promoter control It is anticipated that introduction of enzymes or their homologues will result in increased production of EDA and / or ETrA, respectively, in the transformed host organism. Thus, the present invention converts fatty acid substrates (ie, LA and / or ALA) to the elongase enzymes described herein (eg, EaD9Elo1, EaD9Elo2) such that the substrate is converted to the desired fatty acid product (ie, EDA and / or ETrA, respectively). Or a method of directly producing PUFA comprising exposing to EaD9eS).

More specifically, it is an object of the present invention to provide a method for producing EDA in microbial host cells (eg yeast, algae, bacteria, Euglena, stramenopile, and fungi), ,
(A) a recombinant nucleotide encoding a Δ9 elongase polypeptide having at least 80% amino acid identity based on the Clustal V method of alignment when compared to the polypeptide having the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 A microbial host cell comprising a molecule and (b) a source of LA, wherein the microbial host cell is grown under conditions such that a nucleic acid fragment encoding a Δ9 elongase is expressed and LA is converted to EDA, the EDA optionally recovered Is done.

In an alternative embodiment of the invention, a Δ9 elongase may be used for the conversion of ALA to ETrA. Accordingly, the present invention provides a method of producing ETrA, wherein the microbial host cell is
(A) a recombinant nucleotide encoding a Δ9 elongase polypeptide having at least 80% amino acid identity based on the Clustal V method of alignment when compared to the polypeptide having the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 A microbial host cell comprising a molecule, and (b) a source of ALA, wherein the microbial host cell is grown under conditions such that a nucleic acid fragment encoding a Δ9 elongase is expressed and ALA is converted to ETrA, which is optionally recovered Is done.

Alternatively, each Δ9 elongase gene described herein and its corresponding enzyme product can be used indirectly to produce a variety of ω-6 and ω-3 PUFAs (FIG. 1 and US Pat. No. 7,238). , 482). Indirect production of ω-3 / ω-6 PUFA occurs through an intermediate step or pathway intermediate means in which the fatty acid substrate is indirectly converted to the desired fatty acid product. Thus, enzymes of the PUFA biosynthetic pathway (eg, Δ6 desaturase, C _18/20 elongase, Δ17 desaturase, Δ8 desaturase, Δ15 desaturase, Δ9 desaturase, Δ12 desaturase, C _14/16 elongase, C _16/18 elongase, Δ9 elongase, Δ5 desaturase , [Delta] 4 _desaturase, together with additional genes encoding _{C 20/22} elongase), where Δ9 described elongase (i.e. EaD9Elo1, EaD9Elo2, EaD9eS or other mutant enzymes, codon-optimized enzymes or homologs thereof ) Is expressed resulting in higher level production of longer chain omega-3 / omega-6 fatty acids (eg ARA, EPA, DTA, DPAn-6, DPA and / or DHA) It is considered that it may be.

  In a preferred embodiment, the Δ9 elongase of the invention is (eg, from Euglena gracilis [Wallis et al., Arch. Biochem. And Biophys., 365 (2): 307-316 (May 1999)); No. 2000/34439; US Pat. No. 6,825,017; WO 2004/057001 pamphlet, WO 2006/012325 pamphlet, US Pat. No. 7,256,033, US Patent Application No. 11/635258]; from Acanthamoeba castellani [Sayanova et al., FEBSLett., 580: 1946-1952 (2006)]; Pub [International Publication No. 2005/103253] from Pavlova salina; [International Publication No. 2007/127281] from Pavlova lutheri; Tetreteptopia pomquensis From CCMP1491 [U.S. Patent Application No. 11 / 87,115]; From Eutroptiella sp. CCMP389 [U.S. Patent Application No. 11/876115]; From Utreptiella cf. U.S. Patent Application No. 11/876115]; and Euglena Anavena (Eu) lena Anabaena) [described in co-pending U.S. Patent Application No. 12/099799 specification and U.S. Patent Application No. 12/099811] from) along with Δ8 desaturase will be expressed at a minimum. However, the particular gene contained within a particular expression cassette will depend on the host cell (and its PUFA profile and / or desaturase / elongase profile), substrate availability, and the desired end product.

  In alternative embodiments, the complete sequences described herein, the complements of these complete sequences, a substantial portion of these sequences, codon-optimized elongases derived from them, and substantially homologous thereto Based on this sequence, it may be useful to destroy the natural Δ9 elongase of the host organism.

Microbial expression systems, cassettes, and vectors The Δ9 elongase genes and gene products described herein (ie, EaD9Elo1, EaD9Elo2, EaD9eS or other mutant enzymes, codon-optimized enzymes or homologs thereof) are heterologous microbial hosts It may be expressed in cells, particularly in oleaginous yeast (eg, Yarrowia lipolytica) cells.

  Microbial expression systems and expression vectors containing control sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these can be used to construct a chimeric gene to produce any of the gene products of this sequence. These chimeric genes can then be introduced into suitable microorganisms through transformation to provide high level expression of the encoded enzyme.

  Vectors (eg, constructs, plasmids) and DNA expression cassettes useful for transformation of appropriate microbial host cells are well known in the art. The particular choice of sequences present in the construct will depend on the desired expression product (supra), the nature of the host cell, and the means proposed for separating transformed and non-transformed cells. Typically, however, the vector contains at least one expression cassette, a selectable marker, and sequences that allow autonomous replication or chromosomal integration. Suitable expression cassettes comprise the 5 'region of a gene that controls transcription (eg, a promoter), the gene coding sequence, and the 3' region of a DNA fragment that controls transcription termination (ie, a terminator). Most preferably, both control regions are derived from genes from transformed microbial host cells, but it is understood that such control regions are not necessarily derived from genes native to the particular species selected as the production host. Is done.

  There are many useful transcriptional control regions (also initiation control regions or promoters) that drive the expression of the present Δ9 elongase ORF in the desired microbial host cell and are well known to those skilled in the art. Virtually any promoter capable of inducing the expression of these genes in the selected host cell (ie, natural, synthetic, or chimeric) is suitable for the present invention, but transcription and translation regions from the host species are particularly useful. . Expression in microbial host cells can be achieved in an inducible or constitutive manner. Inducible expression can be achieved by inducing the activity of a controllable promoter that is operably linked to the gene of interest, whereas constitutive expression uses a constitutive promoter that is operably linked to the gene of interest. This can be achieved. By way of example, where the host cell is yeast, transcription and translation regions that are functional in yeast cells, particularly from the host species, are provided (eg for preferred transcription initiation regulatory regions used in Yarrowia lipolytica) See U.S. Patent Application Publication No. 2006-0115881-A1). Any one of several regulatory sequences can be used, depending on whether constitutive or inducible transcription is desired, promoter efficiency in expressing the ORF of interest, ease of construction, etc.

  The nucleotide sequence around the translation initiation codon “ATG” was found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the nucleotide sequence of the foreign gene can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in yeast, this can be done by site-directed mutagenesis by fusing an inefficiently expressed gene in frame to an endogenous yeast gene, which is preferably a highly expressed gene. . Alternatively, consensus translation initiation sequences can be determined in the host and introduced into heterologous genes for their optimal expression in the host of interest.

  The termination region can be derived from the 3 'region of the gene from which the initiation region was derived or from a different gene. A number of termination regions are known and function satisfactorily in a variety of hosts (when used in both the same and different genera and species from which they are derived). The termination region is usually selected for convenience rather than for specific characteristics. Termination control regions may also be derived from various genes native to the preferred host. In alternative embodiments, the 3 'region can also be synthesized, since the skilled person can utilize information available to design and synthesize a 3' region sequence that functions as a transcription terminator. A termination site may be unnecessary in some cases, but is most preferably included.

  As those skilled in the art are aware, simply inserting a gene into a cloning vector does not confirm that it will be successfully expressed at the required level. Many specializations by manipulating several different genetic elements that control aspects of transcription, translation, protein stability, oxygen limitation, and secretion from microbial host cells in response to the need for high expression rates Expression vectors have been created. More specifically, some of the characteristics of molecules that are engineered to control gene expression include the nature of the relevant transcriptional promoter and terminator sequences; the number of copies of the cloned gene (single expression of additional copies) Additional copies may be introduced into the host cell by increasing the plasmid copy number or by multiple integration of the cloned gene into the genome); Whether plasmid-mediated or integrated into the host cell genome; final cellular location of the synthesized foreign protein; translation efficiency in the host organism and precise folding of the protein; mRNA of the clonal gene in the host cell And the inherent stability of the protein; and its frequency is the preferred code of the host cell It includes codon usage in the cloned gene, such as approach frequently used. Each of these types of modifications is encompassed by the present invention as a means of further optimizing the expression of the Δ9 elongase described herein.

Transformation of a microbial host cell Once a suitable DNA cassette (eg, a chimeric gene comprising a promoter, ORF, and terminator) is obtained for expression in a suitable microbial host cell, it can be replicated autonomously in the host cell. Place in a plasmid vector or integrate it directly into the genome of the host cell. The integration of the expression cassette can occur randomly in the host genome, or through the use of a construct containing a region of homology with the host genome sufficient to target genetic recombination at the host locus. Can be determined. If the construct targets an endogenous locus, all or some transcriptional and translational regulatory regions can be provided by the endogenous locus.

  When two or more genes are expressed from separate replicating vectors, each vector should have a different selection means, lacking homology to other constructs, maintaining stable expression, and reconstructing the elements in the construct. Aggregation should be prevented. The judicious selection of regulatory regions, selection means, and introduced construct propagation methods can be determined experimentally so that all introduced genes are expressed at the required level to provide synthesis of the desired product.

  The construct comprising the gene of interest may be introduced into the microbial host cell by any standard technique. These techniques include transformation (eg, lithium acetate transformation [Methods in Enzymology, 194: 186-187 (1991)]), protoplast transformation, ballistic impact, electroporation, microinjection, or gene of interest. Any other method for introducing into a host cell can be mentioned.

  For convenience, host cells that have been engineered in any way that incorporates a DNA sequence (eg, an expression cassette) are referred to herein as “transformed”, “transformants”, or “recombinant”. Accordingly, the terms “transformed” and “recombinant” are used interchangeably herein. The transformed host has at least one copy of the expression construct and may have more than one depending on whether the expression cassette is integrated into the genome or is present on extrachromosomal elements with multiple copies. .

  Transformed host cells are identified by various selection techniques as described in US Pat. No. 7,238,482 and US Pat. No. 7,259,255, and WO 2006/052870. it can.

  Subsequent to transformation, substrates suitable for the present Δ9 elongase (and optionally other PUFA enzymes that are co-expressed in the host cell) may be produced naturally or transgenically by the host, or they may be May be provided exogenously.

Preferred microbial hosts for recombinant expression Microbial host cells for expression of the present genes and nucleic acid fragments include simple or complex carbohydrates, fatty acids, organic acids, oils, glycerol, and alcohols, and over a wide range of temperatures and pH values. And / or hosts that grow on a variety of raw materials, including hydrocarbons. Based on the requirements of the assignees of the applicants, the genes described in the present invention are expressed in oleaginous yeast (especially Yarrowia lipolytica), but the transcription, translation, and protein biosynthesis apparatus. Are highly conserved, so it is contemplated that any bacteria, yeast, algae, Euglena, stramenopile and / or fungus are suitable microbial hosts for expression of the nucleic acid fragment.

  However, preferred microbial hosts are oleaginous organisms such as oleaginous yeast. These organisms can naturally synthesize and accumulate oil, which is greater than about 25% of the cell dry weight, more preferably greater than about 30% of the cell dry weight, and most preferably greater than about 40% of the cell dry weight. Can be configured. The genera typically identified as oleaginous yeasts include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichospomys, Trichospomys However, it is not limited to this. More specifically, exemplary oil synthesizing yeasts include Rhodosporidium toruloides, Lipomyces starkeyii, L. Lipoferas, Candida revkaufi, C.I. Pulcherrima, C.I. Tropicalis, C.I. Utilis, Trichosporon pulls, T. Cutaneum, Rhodotorula glutinus, R. Graminis, and Yarrowia lipolytica (formerly classified as Candida lipolytica). In an alternative embodiment, microbial host cells (eg, yeast) may produce oils in excess of 25% of cell dry weight, so that oil biosynthesis may be genetically modified so that it is considered oily.

  Most preferred is the oleaginous yeast Yarrowia lipolytica, and in yet another embodiment the most preferred are ATCC # 20362, ATCC # 8862, ATCC # 18944, ATCC # 76982 and / or LGAM S (7) 1 Y. It is a lipolytica strain. (Papanikolaou S., and Aggelis G., Bioresource. Technol., 82 (1): 43-9 (2002)).

  Historically, for the production and production of isocitrate lyase, lipase, polyhydroxyalkanoate, citric acid, erythritol, 2-oxoglutaric acid, γ-decalactone, γ-dodecatone, and pyruvic acid, Y. et al. Various strains of lipolytica have been used. Specific teachings applicable to the transformation of oleaginous yeast (ie, Yarrowia lipolytica) include US Pat. No. 4,880,741, US Pat. No. 5,071,764, and Chen. , D.D. C. (Appl. Microbiol. Biotechnol., 48 (2): 232-235 (1997)). Y. Specific teachings applicable to the manipulation of ARA, EPA, and DHA generation in lipolytica are described in US Patent Application No. 11/264784, US Patent Application No. 11 / 257,761, and US Patent Application, respectively. No. 11/264737. The preferred method for expressing genes in this yeast is by integration of linear DNA into the host genome. Integration into multiple locations in the genome can be particularly useful when high level expression of the gene is desired [eg, Ura3 locus (GenBank accession number AJ306421), Leu2 locus (GenBank accession number AF260230), Lys5 locus (GenBank accession number M34929), Aco2 locus (GenBank accession number AJ001300), Pox3 locus (Pox3: GenBank accession number XP — 503244); or Aco3: GenBank accession number AJ001301, US12, desaturase 214,491), Lip1 locus (GenBank accession number Z50020), Lip2 locus (GenBank accession number AJ012632), SCP2 residue Child seat (GenBank Accession No. AJ431362) and / or Pex10 loci into (GenBank Accession No. CAG81606) in.

The preferred selection method used in Yarrowia lipolytica is resistance to kanamycin, hygromycin, and aminoglycoside G418 and the ability to grow in media lacking uracil, leucine, lysine, tryptophan or histidine. In an alternative embodiment, 5-fluoroorotic acid (5-fluorouracil-6-carboxylic acid monohydrate, “5-FOA”) can be used for selection of yeast Ura ^- mutants. The compound is toxic to yeast cells with a functional URA3 gene encoding orotidine 5′-monophosphate decarboxylase (OMP decarboxylase), and therefore based on this toxicity, 5-FOA is a Ura ^- mutant yeast. Particularly useful for strain selection and identification (Bartel, PL and Fields, S., “Yeast 2-Hybrid System”, Oxford University: New York, Vol. 7, 109-147, 1997; (See also WO 2006/052870 for the use of 5-FOA in Yarrowia).

  An alternative preferred selection method used in Yarrowia is based on sulfonylurea (chlorimuron-ethyl from "EI du Pont de Nemours & Co., Inc. (Wilmington, DE)" resistance. Depends on overt non-antibiotic markers from Yarrowia lipolytica. More specifically, the marker gene is a natural acetohydroxyacid synthase (AHAS or acetolactate synthase; EC 4.1.1.3.18) with a single amino acid change (W497L) that confers sulfonylurea herbicide resistance. (International Publication No. 2006/052870 pamphlet). AHAS is the first common enzyme in the branched chain amino acid (ie, valine, leucine, isoleucine) biosynthetic pathway and is the target of sulfonylurea and imidazolinone herbicides.

  Other preferred microbial hosts include oleaginous bacteria, algae, Euglena, stramenopile, and other fungi, of which omega-3 / ω-6 is of particular interest within this broad microbial host population Microorganisms that synthesize fatty acids (or those that can be genetically modified for this purpose [eg other yeasts such as Saccharomyces cerevisiae]). Thus, for example, by transforming Mortierella alpina (used commercially for ARA production) with either this Δ9 elongase gene under the control of an inducible or regulated promoter, Can be transformed into large amounts of DGLA if a Δ8 desaturase gene is co-expressed. M.M. The method of transformation of alpina is described by Mackenzie et al. (Appl. Environ. Microbiol., 66: 4655 (2000)). Similarly, a method for transformation of Thraustochytriales microorganisms (eg, Thraustochytrium, Schizochytrium) is disclosed in US Pat. No. 7,001,772.

  Regardless of the host selected for Δ9 elongase expression described herein, it may be necessary to screen multiple transformants to obtain strains that exhibit the desired expression level and pattern. Such screening includes Southern analysis of DNA blots (Southern, J. Mol. Biol., 98: 503 (1975)), Northern analysis of mRNA expression (Kroczek, J. Chromatogr. Biomed. Appl., 618 (1). ~ 2): 133-145 (1993)), Western and / or Elisa analysis of protein expression, phenotypic analysis or GC analysis of PUFA products.

Based on the above teachings, in one embodiment, the present invention provides:
(A) i) a first recombinant nucleotide molecule encoding a Δ9 elongase polypeptide and operably linked to at least one regulatory sequence; and (ii) an elongase substrate source consisting of LA and / or ALA, respectively. Providing an oleaginous yeast, such as Yarrowia lipolytica;
(B) growing the yeast of step (a) in the presence of a suitable fermentable carbon source to express a gene encoding a Δ9 elongase polypeptide, converting LA to EDA, and / or ALA Converting to ETrA;
(C) relates to a method for producing either EDA or ETrA, comprising the step of optionally collecting EDA and / or ETrA respectively in step (b).

  Substrate supply may be required.

  The nucleotide sequence of the gene encoding the Δ9 elongase may be selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12. In an alternative embodiment, the nucleotide sequence of the gene encoding the Δ9 elongase polypeptide is set forth in SEQ ID NO: 26 (compared to SEQ ID NO: 11 with at least 98 codons expressed in Yarrowia). Have been optimized for).

  PUFAs naturally produced in oleaginous yeast are limited to 18: 2 fatty acids (ie LA) and less commonly 18: 3 fatty acids (ie ALA). In addition to the Δ9 elongase described herein, multiple enzymes required for long-chain PUFA biosynthesis are expressed (thus enabling the production of ARA, EPA, DPA, and DHA, for example).

Specifically, in one embodiment, the present invention provides:
(A) a first recombinant DNA construct comprising an isolated polynucleotide encoding a Δ9 elongase polypeptide and operably linked to at least one regulatory sequence, and (b) a Δ4 desaturase, Δ5 desaturase is selected Δ6 desaturase, [Delta] 9 desaturase, Delta] 12 desaturase, [Delta] 15 desaturase, [Delta] 17 desaturase, [Delta] 8 _{desaturase, C 14/16 elongase, C 16/18 elongase, C 18/20} elongase, and from the group consisting of _{C 20/22} elongase An oleaginous yeast comprising at least one additional recombinant DNA construct comprising an isolated polynucleotide encoding a polypeptide and operably linked to at least one regulatory sequence.

  In a particularly preferred embodiment, at least one additional recombinant DNA construct encodes a polypeptide having Δ8 desaturase activity.

Metabolic engineering of omega-3 and / or omega-6 fatty acid biosynthesis in microorganisms The sequence knowledge of this Δ9 elongase is useful for manipulating omega-3 and / or omega-6 fatty acid biosynthesis in a variety of host cells. Let's go. Methods for manipulating biochemical pathways are well known to those skilled in the art and maximize omega-3 and / or omega-6 fatty acid biosynthesis in oleaginous yeast, particularly in Yarrowia lipolytica. However, it is expected that many operations are possible. This manipulation may require additional engineering of the pathway directly contributing to metabolic engineering within the PUFA biosynthesis pathway or to the biosynthesis pathway from carbon to PUFA. Useful methods for up-regulating desirable biochemical pathways and down-regulating undesirable biochemical pathways are well known to those skilled in the art.

  E.g., biochemical pathways that compete with the ω-3 and / or ω-6 fatty acid biosynthetic pathways for energy or carbon, or natural PUFA biosynthetic pathway enzymes that prevent the production of certain PUFA end products by gene disruption, or It may be down-regulated by other means (eg, antisense mRNA).

  A detailed discussion of operations within the PUFA biosynthetic pathway as a means of increasing ARA, EPA, or DHA (and related techniques) can be found in US Patent Application Publication No. 2006-0094092-A1, US Patent Application Publication No. The same applies to the desired manipulations (and related techniques) within the TAG biosynthetic pathway and the TAG degradation pathway, as in 2006-0115881-A1 and US Patent Application Publication No. 2006-0110806-A1.

  In the context of the present invention, it may be useful to modulate the expression of the fatty acid biosynthetic pathway by any one of the strategies described above. For example, the present invention provides a method for introducing a gene encoding a key enzyme in the Δ9 elongase / Δ8 desaturase biosynthetic pathway into oleaginous yeast for the production of ω-3 and / or ω-6 fatty acids. Various means for metabolic engineering of the host organism are used to express the present Δ9 elongase gene in oleaginous yeast that does not naturally have ω-3 and / or ω-6 fatty acid biosynthetic pathways, It would be particularly useful to modulate gene expression and maximize the production of favorable PUFA products.

Microbial fermentation process for PUFA production Transformed microbial host cells are grown under conditions that optimize expression of the chimeric desaturase and elongase genes to yield the largest and most economical desired PUFA yield . In general, medium conditions that may be optimized include carbon source type and amount, nitrogen source type and amount, carbon-to-nitrogen ratio, amount of different inorganic ions, oxygen level, growth temperature, pH, biomass production Phase length, oil accumulation phase length, and cell harvest time and method. Microorganisms of interest, such as oleaginous yeast (eg, Yarrowia lipolytica) are typically complex media (eg, yeast extract-peptone-dextrose liquid medium (YPD)) or have components necessary for growth. Growing on a synthetic minimal medium (eg, yeast nitrogen-based from DIFCO Laboratories (Detroit, MI)) that lacks selection of the desired expression cassette.

  The fermentation medium in the present invention must contain a suitable carbon source. Suitable carbon sources are taught in US Pat. No. 7,238,482. While it is contemplated that the carbon sources utilized in the present invention may include a wide variety of carbon-containing sources, preferred carbon sources are sugars (eg, glucose), glycerol, and / or fatty acids.

Nitrogen may be supplied from inorganic (eg, (NH ₄ ) ₂ SO ₄ ) or organic (eg, urea or glutamic acid) sources. In addition to appropriate carbon and nitrogen sources, the fermentation medium is also suitable for the growth of appropriate minerals, salts, cofactors, buffers, vitamins, and oily hosts, and for promoting the enzymatic pathways essential for PUFA production. It must contain other components known to those skilled in the art. Several metal ions (eg Fe ⁺² , Cu ⁺² , Mn ⁺² , Co ⁺² , Zn ⁺² , Mg ⁺² ) that promote the synthesis of lipids and PUFAs are of interest (edited by DJ Kyle and R. Colin, From “Ind. Appl. Single Cell Oil”, Nakahara, T. et al., 61-97 (1992).

  The preferred growth medium in the present invention is a common commercial conditioned medium such as yeast nitrogen base from DIFCO Laboratories (Detroit, MI). Other synthetic or artificial growth media may also be used, and media suitable for the growth of transformed host cells are known to those skilled in the microbiology or fermentation science arts. Suitable pH ranges for fermentation are typically between about pH 4.0 and pH 8.0, with pH 5.5 to pH 7.5 being preferred as the range of initial growth conditions. Fermentation may be carried out under aerobic or aerobic conditions, with microaerobic conditions being preferred.

  Typically, high levels of PUFA accumulation in oleaginous yeast cells require a two-step process because the metabolic state must be "equilibrium" between growth and adipose synthesis / storage. Thus, most preferably, PUFA production in oleaginous yeast (eg, Yarrowia lipolytica) requires a two-stage fermentation process. This approach is described in US Pat. No. 7,238,482, as well as various suitable fermentation process designs (ie, batch, fed batch, and continuous) and growing considerations.

Purification and processing of PUFA oils PUFAs may be included in the host microorganism as free fatty acids or in esterified forms such as acylglycerols, phospholipids, sulpholipids or glycolipids and are well known in the art. It may be extracted from the host cell through any means. A review of yeast lipid extraction techniques, quality analysis, and acceptance criteria can be found in Z. Jacobs (Critical Reviews in Biotechnology, 12 (5/6): 463-491 (1992)). A. Singh and O. A short review of the post-treatment process by Ward (Adv. Appl. Microbiol., 45: 271-312 (1997)) is also available.

  In general, means for purifying PUFAs include extraction with organic solvents (eg, US Pat. No. 6,797,303 and US Pat. No. 5,648,564), sonication (eg, using carbon dioxide). ) Physical means such as supercritical fluid extraction, saponification, and pressing, or combinations thereof may be included. For further details, see the teachings of US Pat. No. 7,238,482.

PUFA-containing oils for use in foodstuffs, health foods, pharmaceuticals, and animal feeds The market currently incorporates omega-3 and / or omega-6 fatty acids (eg in particular ALA, GLA, ARA, EPA, DPA, and DHA) Support a wide variety of food and feed products. Microbial resources comprising long chain PUFAs, partially purified microbial resources comprising PUFAs, purified microbial oils comprising PUFAs, and / or purified PUFAs are food and feed products It is contemplated that it will function within and provide the health benefits of the formulation. More specifically, the oil of the present invention containing omega-3 and / or omega-6 fatty acids includes similar foods, meat products, cereal products, baked foods, snack foods, and dairy products. Suitable for use in a variety of food and feed products including but not limited to (see US 2006/0094092 for details).

  In addition, the compositions may be used in formulations to provide health benefits to medical foods, including medical nutrition, nutritional supplements, infant formula and pharmaceuticals. Those skilled in the art of food processing and formulating will understand how the amount and composition of the oil may be added to the food or feed product. Such an amount is referred to herein as an “effective” amount and is intended to correct or treat a food or feed product, a diet or medical food that the product is intended to supplement, or a medical nutrition It depends on the disease.

  Unless otherwise stated, the invention is further defined in the following examples, where parts and percentages are by weight and temperatures are in degrees Celsius. These examples are provided for illustrative purposes only, while illustrating preferred embodiments of the invention. From the above considerations and these examples, those skilled in the art will be able to ascertain the essential characteristics of the invention and make various changes and modifications to the invention without departing from its spirit and scope. And can meet the requirements. Accordingly, various modifications of the invention in addition to those presented and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

General Methods Standard recombinant DNA and molecular cloning techniques used in the examples are well known in the art, and ) Sambrook, J .; Fritsch, E .; F. And Maniatis, T .; “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989) (Maniatis); ) T. J. et al. Silhavy, M.M. L. Bennan, and L.M. W. 2. Enquist, “Experiments with Gene Fusions”, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1984); ) Ausubel, F.M. M.M. Et al., “Current Protocols in Molecular Biology”, Green Publishing Assoc. and published by Wiley-Interscience (1987).

  Suitable materials and methods for maintaining and growing microbial cultures are well known in the art. Techniques suitable for use in the following examples are described below. Phillip Gerhardt, R.C. G. E. Murray, Ralph N .; Costilow, Eugene W. Nester, Willis A. Wood, Noel R.D. Krieg, and G.K. Edited by Briggs Phillips, “Manual of Methods for General Bacteriology”, American Society for Microbiology, Washington, D .; C. (1994), or Thomas, D. et al. Block, “Biotechnology: A Textbook of Industrial Microbiology”, 2nd edition, Sinauer Associates: Sunland, MA (1989). All reagent restriction enzymes and materials used for the growth and maintenance of microbial cells are Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO / BRL (Gaithersburg, MD) unless otherwise noted. ), Or from Sigma Chemical Company (St. Louis, MO). E. coli (XL1-Blue) competent cells were purchased from Stratagene Company (San Diego, Calif.). E. coli strains were typically grown at 37 ° C. on Luria Bertani (LB) plates.

  General molecular cloning was performed according to standard methods (Sambrook et al., Supra). DNA sequences can be obtained using ABI using dye terminator technology (US Pat. No. 5,366,860, EP 272,007) using a combination of vector and insert-specific primers. Generated on an automatic sequencer. Sequence editing was performed in a Sequencher from “Gene Codes Corporation (Ann Arbor, MI)”. All sequences represent at least twice coverage in both directions. For comparison of gene sequences, see DNASTAR Inc. Achieved using DNASTAR software from (Madison, WI).

  The meanings of the abbreviations are as follows. “Sec” means seconds, “min” means minutes, “h” or “hr” means hours, “d” means days, “μL” means microliters, “ML” means milliliter, “L” means liter, “μM” means micromolar, “mM” means millimolar, “M” means molar, “mmol” means millimole, “μmole” means micromole, “g” means gram, “μg” means microgram, “ng” means nanogram, “U” means Unit means "bp" means base pair and "kB" means kilobase.

Expression cassette nomenclature:
The expression cassette structure is represented by a simple notation system of “X :: Y :: Z”, where X represents a promoter fragment, Y represents a gene fragment, and Z represents a terminator fragment, all of which are operatively linked to each other. To do.

Transformation and culture of Yarrowia lipolytica:
Yarrowia lipolytica strains with ATCC registration numbers # 20362, # 76982, and # 90812 were purchased from the US Microbial Strains (Rockville, MD). Yarrowia lipolytica strains were typically grown at 28-30 ° C. in several media according to the recipe shown below. Agar plates were prepared as needed by adding 20 g / L agar to each liquid medium according to standard methods.

  YPD agar (per liter): 10 g yeast extract [Difco], 20 g Bacto peptone [Difco], and 20 g glucose.

  Basal minimal medium (MM) (per liter): 20 g glucose, 1.7 g amino acid free yeast nitrogen base, 1.0 g proline, pH 6.1 (no regulation).

  Minimal medium + 5-fluoroorotic acid (MM + 5-FOA) (per liter): 20 g glucose, 6.7 g yeast nitrogen base, 75 mg uracil, 75 mg uridine, and variations within each batch received from the supplier A suitable amount of Zymo Research Corp. based on FOA activity tested for a range of concentrations from 100 mg / L to 1000 mg / L. FOA from (Orange, CA).

  Transformation of Yarrowia lipolytica was carried out by Chen, D. et al. Unless otherwise noted. C. (Appl. Microbiol. Biotechnol., 48 (2): 232-235 (1997)). Briefly, Yarrowia was streaked on YPD plates and grown at 30 ° C. for approximately 18 hours. Several heaps of platinum loops of cells were scraped from the plate and resuspended in 1 mL of transformation buffer comprising: 2.25 mL 50% PEG, average MW 3350; 0.125 mL 2M lithium acetate, pH 6.0; 0.125 mL 2MDTT; and (optionally) 50 μg sheared salmon sperm DNA. Approximately 500 ng of linear DNA (or preferably 100 ng circular plasmid), preferably comprising at least one chimeric gene, is then incubated in 100 μL of resuspended cells with vortex mixing at 15 min intervals It was kept at 39 ° C. for 1 hour. Cells were seeded on selective media plates and kept at 30 ° C. for 2-3 days.

Fatty acid analysis of Yarrowia lipolytica For fatty acid analysis, Bligh, E., et al. G. And Dyer, W .; J. et al. Can. J. et al. Biochem. Physiol. 37: 911-917 (1959), cells were collected by centrifugation and lipids extracted. Fatty acid methyl esters were prepared by transesterification of lipid extracts with sodium methoxide (Roughhan, G. and Nishida, I., Arch Biochem Biophys. 276 (1): 38-46 (1990)), followed by Analysis was performed on a Hewlett-Packard 6890 GC equipped with a 30 m x 0.25 mm (inner diameter) HP-INNOWAX column from Hewlett-Packard. The oven temperature was 3.5 ° C./min, 170 ° C. (25 minutes hold) to 185 ° C.

  For direct base transesterification, a Yarrowia culture (3 mL) was collected, washed once with distilled water, and dried under vacuum in a Speed-Vac for 5-10 minutes. Sodium methoxide (100 μL of 1%) was added to the sample, and then the sample was vortexed and shaken for 20 minutes. After adding 3 drops of 1M NaCl and 400 μL of hexane, the sample was vortexed and centrifuged. The upper layer was removed and analyzed by GC as described above.

Example 1
Synthesis of cDNA library from Euglena anabena UTEX 373 This example describes the synthesis of cDNA library from Euglena anabena UTEX 373. This work included RNA preparation, cDNA synthesis, and creation of a cDNA library.

Growth and RNA preparation of Euglena anabeena UTEX 373 Euglena anabeena UTEX 373 was obtained from Dr. Richard Trimer's laboratory, Dr. Richard Trimer, East Lansing, MI. Approximately 2 mL of culture was removed for lipid analysis and centrifuged at 1,800 × g for 5 minutes. The pellet was washed once with water and centrifuged again. The resulting pellet was dried under vacuum for 5 minutes, resuspended in 100 μL trimethylsulfonium hydroxide (TMSH) and incubated for 15 minutes at room temperature with shaking. After incubation, 0.5 mL of hexane was added and the vials were further incubated for 15 minutes at room temperature with shaking. Fatty acid methyl esters (5 μL injected from the hexane layer) were separated and quantified using a Hewlett-Packard 6890 gas chromatograph equipped with an Omegawax 320 fused silica capillary column (Supelco Inc., catalog number 24152). The oven temperature was programmed and maintained at 170 ° C. for 1.0 minute, increased to 240 ° C. at 5 ° C./minute, and then maintained for an additional 1.0 minute. The carrier gas was supplied by a Whatman hydrogen generator. FIG. 2 shows the chromatogram obtained by comparing the residence time with a commercially available standard methyl ester (Nu-Chek Prep, Inc., catalog number U-99-A). The presence of EDA, ETrA, EPA, and DHA in the fatty acid profile, along with the absence of GLA and STA, Euglena anabena uses the Δ9 elongase / Δ8 desaturase pathway for long chain (LC) PUFA biosynthesis And suggested that it can be a good source of LC-PUFA biosynthetic genes, including but not limited to Δ9 elongases.

  The remaining 5 mL of vigorously growing culture was added to 25 mL AF-6 medium (Watanabe & Hiroki, NIES-Collection List of Strains, 5th edition, National Institute for Environmental Studies, 4th year, Tokyo University, 4th grade, 200th grade). ) Euglena anabena cultures were grown at 22 ° C. for 2 weeks with a cycle of 16 hours light and 8 hours dark with very gentle agitation.

After 2 weeks, the culture (25 mL) was transferred to 100 mL AF-6 medium in a 500 mL glass bottle and the culture was grown for 1 month as described above. After this period, two 50 mL aliquots were transferred into two separate 500 mL glass bottles containing 250 mL AF-6 medium and the cultures were grown for 2 months as described above (a total of about 600 mL of culture was Obtained). The culture was then pelleted by centrifugation at 1,800 × g for 10 minutes, washed once with water and centrifuged again. TEL-TEST, Inc. Using the RNA STAT-60 ^™ reagent from (Friendswood, TX) and following the provided manufacturer's protocol (dissolve RNA in 0.5 mL water using 5 mL reagent) Total RNA was extracted from one. In this way, 340 μg of total RNA (680 μg / mL) was obtained from the pellet. The remaining pellet was frozen in liquid nitrogen and stored at -80 ° C. MRNA was isolated from all 340 μg of total RNA according to the manufacturer's protocol provided using the mRNA purification kit from Amersham Biosciences (Piscataway, NJ), thus obtaining 9.0 μg of mRNA. It was.

Preparation of Euglena anabena cDNA and creation of cDNA library eug1c Using the Clonemin ^™ cDNA library construction kit (Cat # 18249-029) from Invitrogen Corporation (Carlsbad, Calif.) A cDNA library was prepared according to the protocol (version B, 25-0608). Using non-radiolabeling method, cDNA was synthesized from 5.12 μg of mRNA (described above) using biotin-attB2-oligo (dT) primer. After synthesis of the first and second strands, the attB1 adapter was added and ligated, and the cDNA was size fractionated using column chromatography. DNA from the fractions was concentrated, genetically modified into pDONR ^™ 222 and transformed into E. coli ElectroMAX ^™ DH10B ^™ T1 phage-resistant cells (Invitrogen Corporation). The Euglena anabena library was named eug1c.

The cDNA library eug1c was seeded on LB + kanamycin plates (approximately 100,000 colonies), and the colonies were scraped off. Qiagen Inc. (Valencia, CA) and DNA was isolated according to QIAprep ^(R) Spin Miniprep Kit using the manufacturers protocol from. In this way, a plasmid DNA sub-library from eug1c was obtained.

Example 2
Isolation of full-length Δ9 elongase from Euglena anabena UTEX 373 This example describes the identification of a cDNA (SEQ ID NO: 1 and 2) encoding a Δ9 elongase from Euglena anabena UTEX 373. ing. This study was made of probes derived from Euglena gracilis Δ9 elongase (EgD9e; SEQ ID NO: 3), and a probe and cDNA library to identify Δ9 elongase homologues from Euglena anabena UTEX 373 Hybridization with eug1c was included.

Euglena gracilis Δ9 elongase (EgD9e)
New England Biolabs Inc. Euglena gracilis Δ9 elongase (EgD9e; SEQ ID NO: 3; US Patent Application No. 11 / 601,563) according to the manufacturer's protocol using VentR® DNA polymerase (Cat. No. M0254S) from (Beverly, MA). As described in the specification). pk001. EgD9e was amplified with oligonucleotide primers oEugEL1-1 (SEQ ID NO: 5) and oEugEL1-2 (SEQ ID NO: 6) using a clone (egeg1c) from the Euglena cDNA library called n5f as a template. . The resulting DNA fragment was cloned into the pCR-Blunt® cloning vector using the Zero Blunt® PCR cloning kit (Invitrogen Corporation) according to the manufacturer's protocol to generate pKR906 (SEQ ID NO: 15) .

Colony Lift Three large square (24 cm × 24 cm) Petri dishes from Corning (Corning, NY), each containing LB + 50 μg / mL kanamycin agar medium, approximately 17 of the Euglena anabenaa cDNA library eug1c. 000 clones were seeded. Cells were grown overnight at 37 ° C. and then the plates were cooled to room temperature.

  A Biodyne B 0.45 μm membrane (Catalog No. 60207) from Pall Corporation (Pensacola, FL) was cut to approximately 22 cm × 22 cm and the membrane was carefully placed on agar avoiding air bubbles. After 2 minutes incubation at room temperature, the membrane orientation was labeled and lifted with forceps and placed on a filter paper soaked in 0.5 M sodium hydroxide and 1.5 M sodium chloride with the colony side up. After denaturation for 4 minutes, the membrane was placed on a filter paper soaked in 0.5 M Tris-HCL (pH 7.5) and 1.5 M sodium chloride for 4 minutes to neutralize sodium hydroxide. The steps were repeated and the membrane was quickly rinsed in 2x SSC buffer (20x SSC is 3M sodium chloride, 0.3M sodium citrate, pH 7.0) and air dried on filter paper.

Hybridization Membranes were prehybridized in 200 mL hybridization solution for 2 hours at 65 ° C. The hybridization solution was 6 × SSPE (20 × SSPE is 3M sodium chloride, 0.2M sodium phosphate, 20 mM EDTA, pH 7.4), 5 × Denhardt's reagent (100 × Denhardt's reagent is 2% (w / v) Ficoll. 2% (w / v) polyvinylpyrrolidone, 2% (w / v) acetylated bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS), 100 μg / mL sheared salmon sperm DNA, and 5% dextran sulfate. Contained.

P ³² using an agarose gel purified NcoI / NotI DNA fragment containing the Δ9 elongase gene using the RadPrime DNA labeling system from Invitrogen (Carlsbad, Calif.) (Cat. No. 18428-011) according to the manufacturer's instructions. Euglena gracilis DNA probe was prepared from pKR906 (SEQ ID NO: 15) labeled with dCTP. Unincorporated P ³² dCTP was separated using a NICK column (catalog number 17-0855-02) from Amersham Biosciences (Piscataway, NJ) according to the manufacturer's instructions. The probe was denatured at 100 ° C. for 5 minutes, placed on ice for 3 minutes, and half was added to the hybridization solution.

  Hybridize the membrane with the probe overnight at 65 ° C. with gentle shaking, then the next day twice in 2 × SSC containing 0.5% SDS (5 minutes each) with 0.1% SDS. Washed twice with contained 0.2 × SSC (15 minutes each). After washing, the hyperfilm (Catalog Number RPN30K) from Amersham Biosciences (Piscataway, NJ) was exposed to the membrane at -80 ° C overnight.

  Based on the alignment of the exposed hyperfilm and the plate, positive colonies were picked into 1 mL of water using a blunt end of a Pasteur pipette and vortexed. Several dilutions were made and seeded on small circular Petri dishes (82 mm) containing LB medium supplemented with 50 μg / mL kanamycin, resulting in about 100 well-isolated colonies on a single plate. Hybridization was performed in 100 mL using the remaining radiolabeled probe, lifted as described above, except that the Nytran N membrane circle (Catalog # 10416116) from Schleicher & Schuell (Keene, NH) was used. In this way, positive clones were confirmed.

  Individual positive clones were grown in LB + 50 μg / mL kanamycin liquid medium at 37 ° C. and plasmids were purified using the QIAprep® Spin Miniprep kit (Qiagen Inc.) according to the manufacturer's protocol.

  Using the ABI BigDye version 3 Prism sequencing kit, the vector-stimulated M13F universal primer (SEQ ID NO: 7), the M13rev-28 primer (SEQ ID NO: 8), and the Poly (A) tail pre-stimulated WobbleT oligo Nucleotides were used to end-sequence the DNA insert in a 384 well plate. For the sequencing reaction, the following reaction conditions were repeated 25 times using 100-200 ng template and 6.4 pmol primer. 96 ° C for 10 seconds, 50 ° C for 5 seconds, and 60 ° C for 4 minutes. After ethanol-based purification, cycle sequencing reaction products were analyzed and detected on a Perkin-Elmer ABI 3700 automated sequencer. The WobbleT primer is an equimolar mixture of 21mer poly (T) A, poly (T) C, and poly (T) G used for sequencing the 3 'end of the cDNA clone.

The clones are referred to as two different groups (EaD9Elo1 and EaD9Elo2) based on the insert sequence by aligning and comparing the sequences using Sequencher ^™ (version 4.2) from Gene Codes Corporation (AnnArbor, MI). ). Representative clones containing cDNA for each sequence class were selected for further study, and the sequence of each representative plasmid (ie, pLF121-1 and pLF121-2) is shown by SEQ ID NO: 9 and SEQ ID NO: 10, respectively. The sequence indicated by the NNNN string corresponds to the region of the polyA tail that was not sequenced. The coding sequences for EaD9Elo1 and EaD9Elo2 are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively. The amino acid sequences corresponding to EaD9Elo1 and EaD9Elo2 are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

Example 3
Primary sequence analysis of Euglena anabeena UTEX 373 (EaD9Elo1 and EaD9Elo2) Δ9 elongase sequence and comparison with other published Δ9 elongase sequences DNASTAR Inc. LASERGENE Bioinformatics Computation Suite MegAlign ^™ v6.1 program from (Madison, WI) default parameters for pair-wise alignment (KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5 and DIAGONALS SAVED = 5 and GAP LENTHTHPE ) And the Clustal V method (Higgins, DG and Sharp, PM, Comput. Appl. Biosci., 5: 151-153 (1989); Higgins et al., Comput. Appl. Biosci. 8: 189-191 (1992)) using EaD9Elo1 (SEQ ID NO: 13) and EaD9Elo2 (SEQ ID NO: 14) amino acids. The no acid sequences were compared.

  Compared to EaD9Elo1 (SEQ ID NO: 13), EaD9Elo2 (SEQ ID NO: 14) has one amino acid substitution (ie R254Q; relative to the numbering of EaD9Elo1). The nucleotide sequences of EaD9Elo1 (SEQ ID NO: 11) and EaD9Elo2 (SEQ ID NO: 12) differ by 6 base pairs over the full length of 774 bp.

  In the BLAST “nr” database (comprising all non-redundant GenBank CDS translations and the three-dimensional structure Brookhaven protein data bank, the latest major release of the SWISS-PROT protein sequence database, sequences from the EMBL and DDBJ databases) BLASTP (Basic Local Alignment Search Tool) searching for similarity to contained sequences; Altschul et al., J. Biol. Mol. Biol. 215: 403-410 (1993)) with the default parameters turned off and the amino acid sequences of EaD9Elo1 (SEQ ID NO: 13) and EaD9Elo2 (SEQ ID NO: 14) were evaluated. For convenience, the P value (probability) at which the match between the cDNA sequence and the sequence contained in the searched database is observed only by chance in the calculation by BLAST corresponds to the negative number of the logarithm of the P value reported here. Report as "pLog" value. Therefore, the larger the pLog value, the greater the likelihood that the cDNA sequence and BLAST “hit” represent homologous proteins.

  Compared to the “nr” database, both sequences are Isochrysis galvana long chain polyunsaturated fatty acid elongation enzyme (IgD9e; SEQ ID NO: 16) (NCBI accession number AAL37626 (GI17226123), locus AAL37626, CDSAF390174; Qi et al., FEBS Lett., 510: 159-165 (2002)) resulted in a pLog value of 38.70 (P value of 2e-39). The BLAST score and probability suggests that the nucleic acid fragment encodes the entire Euglena anabena Δ9 fatty acid elongase.

  Using the BlastP, Clustal V and Jotun Hein methods of sequence comparison, the amino acid sequence of EaD9Elo1 (SEQ ID NO: 13) and EaD9Elo2 (SEQ ID NO: 14) is changed to IgD9e (SEQ ID NO: 16) and Euglena gracilis Δ9 elongase amino acid sequence. (EgD9e; SEQ ID NO: 4; WO 2007/061845 pamphlet). The percent identity to IgD9e and EgD9e using each method is shown in Table 4 and Table 5, respectively.

The percent sequence identity was calculated by the BlastP and Clustal V methods as described above. The sequence% identity calculations performed by the Jotun Hein method (Hein, JJ, Meth. Enz, 183: 626-645 (1990)) are reported in DNASTAR Inc. The LASERGENE bioinformatics suite MegAlign ^™ v6.1 program from (Madison, Wis.) Was used with the default parameters for pairwise alignment (KTUPLE = 2).

Example 4
Functional analysis of Euglena anabeena UTEX 373Δ9 elongase in Yarrowia lipolytica (Earlo number 9 in Yarrowia lipolytica) The functional analysis is described. The study consists of (1) constructing a Gateway® compatible Yarrowia expression vector pY159, (2) transferring EaD9Elo1 and EaD9Elo2 to pY159 to generate pY173 and pY174, (3) comparing lipid profiles in transformed organisms comprising pY173 and pY174.

Construction of Gateway® Compatible Yarrowia Expression Vector pY159 Plasmid pY5-30 (as already described in US Pat. No. 7,259,255) is used for E. coli and Yarrowia. A shuttle plasmid that can replicate in both Yarrowia lipolytica. Plasmid pY5-30 is selected from the Yarrowia autonomously replicating sequence (ARS18), the ColE1 plasmid origin of replication, the ampicillin resistance gene for selection in E. coli (Amp ^R ), and the selection in Yarrowia. Contains the Yarrowia LEU2 gene for and the chimeric TEF :: GUS :: XPR gene. Using techniques well known to those skilled in the art, the TEF promoter is replaced with the Yarrowia lipolytica FBAINm promoter (US Pat. No. 7,202,356) to obtain plasmid pDMW263 (SEQ ID NO: 17 ) Was produced from pY5-30. Briefly, this promoter is located in the 5 ′ upstream untranslated region before the “ATG” translation initiation codon of the fructose-bisphosphate aldolase enzyme (EC 4.1.1.23) encoded by the fba1 gene. FBAINm is a 52 bp deletion between the ATG translation initiation codon of the FBAIN promoter and the intron (resulting in 22 N-terminal 22 fragments). And a new translational consensus motif after the intron. Table 6 summarizes the components of pDMW263 (SEQ ID NO: 17).

  Contains a synthetic Δ9 elongase gene (IgD9eS) derived from Isochrysis galvana and codon-optimized for expression in Yarrowia lipolytica, the contents of which are hereby incorporated by reference. From pDMW263 (SEQ ID NO: 17) containing the Yarrowia lipolytica FBAINm promoter to the NcoI / SalI DNA fragment of pDMW237 (SEQ ID NO: 18) previously described in WO 2006/012325. The NcoI / SalI DNA fragment of was cloned to generate pY115 (SEQ ID NO: 19; FIG. 3A). In FIG. 3A, the modified FBAINm promoter is labeled as FBA1 + intron, whereas in FIGS. 3B, 3C, and 3D, it is labeled as YAR FBA1 PRO + intron.

  The FBAINm promoter was amplified from plasmid pY115 (SEQ ID NO: 19) using PCR with oligonucleotide primers oYFBA1 (SEQ ID NO: 20) and oYFBA1-6 (SEQ ID NO: 21). Primer oYFBA1 (SEQ ID NO: 20) is designed to introduce a BglII site at the 5 ′ end of the promoter, and primer oYFBA1-6 (SEQ ID NO: 21) is located at the 3 ′ end of the promoter while removing the NcoI site and thus the ATG start codon. Designed to introduce a NotI site. The resulting PCR fragment was digested with BglII and NotI and cloned into the BglII / NotI fragment of pY115 containing the vector backbone to form pY158 (SEQ ID NO: 22).

  Plasmid pY158 (SEQ ID NO: 22) was digested with NotI and the resulting DNA ends were filled. After filling to form blunt ends, the DNA fragments were treated with calf intestinal alkaline phosphatase and separated using agarose gel electrophoresis. A 6992 bp fragment containing the Yarrowia lipolytica FBAINm promoter was excised from the agarose gel, and Qiagen Inc. Purified using the QIAquick® gel extraction kit from (Valencia, CA) according to the manufacturer's protocol. The purified 6992 bp fragment was ligated with cassette rfA using the Gateway vector conversion system (Cat. No. 11823-029, Invitrogen Corporation) according to the manufacturer's protocol, and the Yarrowia lipolytica Gateway target vector. pY159 (SEQ ID NO: 23; FIG. 3B) was formed.

Construction of Yarrowia expression vectors pY173 and pY174 pLF121-1 (SEQ ID NO: 9; implementation using the Gateway® LR Clonase ^™ II enzyme mix (Catalog No. 11791-020, Invitrogen Corporation) according to the manufacturer's protocol The cDNA inserts from Example 2) and pLF121-2 (SEQ ID NO: 10; Example 2) were transferred to pY159 (SEQ ID NO: 23), and pY173 (SEQ ID NO: 24; FIG. 3C) and pY174 (SEQ ID NO: 25; FIG. 3D) was formed respectively.

Functional analysis of EaD9Elo1 and EaD9Elo2 in Yarrowia lipolytica Y2224 strain Y2224 strain was isolated as follows. YPD agar on MM plates (75 mg / L uracil and uridine each, 6.7 g / L YNB with no ammonium sulfate, no amino acids, 20 g / L glucose) containing 250 mg / L 5-FOA (Zymo Research) Yarrowia lipolytica ATCC # 20362 cells from the plate (1% yeast extract, 2% bactopeptone, 2% glucose, 2% agar) were streaked. Plates were incubated at 28 ° C. and 4 of the resulting colonies were separately patch-cultured on MM plates containing 200 mg / mL 5-FOA and MM plates lacking uracil and uridine to obtain uracil Ura3 Nutritional requirements were confirmed.

  The Y2224 strain was transformed with pY173 (SEQ ID NO: 24; FIG. 3C) and pY174 (SEQ ID NO: 25; FIG. 3D) as described in the general method.

  Single colonies of transformed Yarrowia lipolytica containing pY173 and pY174 were grown in 3 mL of MM lacking uracil for 16 hours at 30 ° C., after which the cells were pelleted by centrifugation at 250 rpm. Cells were washed once with water, pelleted by centrifugation and air dried. Transesterify the pellet with 500 μL of 1% sodium methoxide at 50 ° C. for 30 min (Roughhan, G. and Nishida, I., Arch. Biochem. Biophys., 276 (1): 38-46 (1990) ), Then 500 μL of 1M sodium chloride and 100 μL of heptane were added. After thorough mixing and centrifugation, fatty acid methyl esters (FAME) were analyzed by GC. FAME (5 μL injected from the hexane layer) was separated and quantified using a Hewlett-Packard 6890 gas chromatograph equipped with an Omegawax 320 fused silica capillary column (Catalog No. 24152, Supelco Inc.). The oven temperature was programmed and held at 220 ° C. for 2.6 minutes, increased to 240 ° C. at 20 ° C./min, and then held for another 2.4 minutes. The carrier gas was supplied by a Whatman hydrogen generator. Residence times were compared with commercially available standard methyl esters (Nu-Chek Prep, Inc.).

  The fatty acid profile of Yarrowia lipolytica expressing pY173 and pY174 is shown in Table 7. Fatty acids are 16: 0, 16: 1, 18: 0, 18: 1 (oleic acid), LA, 20: 0, 20: 1 (11), EDA, 22: 0, 24: 0, and 24: 1 Identified. % Δ9 Elongation (Δ9% Elong) was calculated by dividing the weight parts (% by weight) of EDA by the sum of the weight percentages of EDA and LA and multiplying by 100 to express as%. The average is Ave. It is represented by

Example 5
Synthesis of a codon-optimized Δ9 elongase gene (EaD9ES) for Yarrowia lipolytica as described in WO 2004/101753 and US Pat. No. 7,125,672. In a similar manner, the codon usage of the Euglena anabena Δ9 elongase gene (EaD9Elo1) was optimized for expression in Yarrowia lipolytica. Specifically, a codon-optimized Δ9 elongase gene (referred to as “EaD9ES”; SEQ ID NO: 26) is based on the coding sequence of EaD9Elo1 (SEQ ID NO: 11) and the Yarrowia codon usage pattern (international (Publication No. 2004/101753), consensus sequence around the "ATG" translation initiation codon, and the principle of RNA stability (Guhaniyogi, G. and J. Brewer, Gene, 265 (1-2): 11-23 (2001). Designed according to year)). In addition to modification of the translation start site, 106 bp (13.7%) of the 774 bp coding region was modified to optimize 98 codons (38.0%). The GC content (52.1%) was almost identical between the wild type gene (ie EaD9Elo1) and the synthetic gene (ie EaD9ES). NcoI and NotI sites were incorporated around the translation start codon and after the stop codon of EaD9ES (SEQ ID NO: 26), respectively. 4A and 4B show a comparison of the nucleotide sequences of EaD9Elo1 (SEQ ID NO: 11) and EaD9ES (SEQ ID NO: 26). The protein sequence (ie, SEQ ID NO: 27) encoded by the codon optimized gene is identical to the wild-type protein sequence (ie, SEQ ID NO: 13). The designed EaD9ES gene was synthesized by GenScript Corporation (Piscataway, NJ) and cloned into pUC57 (Genbank accession number Y14837) to create pEaD9ES (SEQ ID NO: 28; FIG. 5A).

Example 6
Yarrowia lipolytica expression comprising a synthetic Δ9 elongase gene (EaD9ES) (derived from Euglena anabeena) codon-optimized for expression in Yarrowia lipolytica (Yarrowia lipolytica) Construction and Functional Analysis of Vector pZUFmEaD9ES This example describes the functional expression of the Yarrowia lipolytica vector pZUFmEaD9ES comprising the chimeric FBAINm :: EaD9ES :: Pex20 gene, and EaD9ES is a Euglena derived from anabaena) and originated in Yarrowia A codon optimized synthetic Δ9 elongase for. Plasmid pZUFmEaD9ES (FIG. 5B) contained the following components:

Functional analysis of Yarrowia lipolytica transformants comprising pZUFmEaD9ES As described in General Methods, plasmid pZUFmEaD9ES was transformed into the Y2224 strain (abrupt Ura3 gene autonomous of the wild type Yarrowia ATCC # 20362 strain). FOA resistant mutants from mutations). Transformants were selected on the top MM plate. After growth at 30 ° C. for 2 days, the transformant was picked up and streaked again on a fresh MM plate. Once grown, these strains were individually inoculated into 3 mL liquid MM at 30 ° C. and shaken at 250 rpm / min for 2 days. Cells were harvested by centrifugation to extract lipids, fatty acid methyl esters were prepared by transesterification and subsequently analyzed on a Hewlett-Packard 6890 GC.
GC analysis showed that approximately 2.2% of total lipids C20: 2 (EDA) and 15.3% C18: 2 (LA) were produced in all 5 transformants, among these 5 strains. The conversion efficiency from C18: 2 to C20: 2 was determined to be about 13%. Therefore, this experimental data shows that a synthetic Euglena anabaena Δ9 elongase (ie, EaD9ES described in SEQ ID NOs: 26 and 27), codon optimized for expression in Yarrowia lipolytica, It has been demonstrated to actively extend LA to EDA.

The present invention is summarized as follows.
1. (A) a nucleotide having Δ9 elongase activity and encoding a polypeptide having at least 80% amino acid identity based on the clustered V method of alignment when compared to the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 Array,
(B) encodes a polypeptide having Δ9 elongase activity and, when compared to the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 26, at least 80% sequence identity based on the blast N method of alignment Having a nucleotide sequence,
(C) a nucleotide sequence encoding a polypeptide having Δ9 elongase activity and hybridizing under stringent conditions to the nucleotide sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 26, or (d) (a ), A microbial host cell comprising an isolated polynucleotide comprising the complement of the nucleotide sequence of (b) or (c), the complement and the nucleotide sequence comprising the same number of nucleotides and being 100% complementary.
2. 2. The microbial host cell according to 1 above, wherein the isolated polynucleotide encodes the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14.
3. 2. The microbial host cell according to 1 above, which is selected from the group consisting of yeast, algae, bacteria, Euglena, stramenopile, and fungi.
4). 4. The microbial host cell according to 3 above, wherein the cell is a fungus belonging to the genus Penicillium.
5. 4. The microbial host cell according to 3 above, wherein the cell is a stramenopile selected from the group consisting of a species of Amaranthus species and Schizochytrium species.
6). 4. The microbial host cell according to 3 above, wherein the yeast is oleaginous yeast.
7). 7. The microbial host cell according to 6 above, wherein the oleaginous yeast is selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichospolone, and Lipomyces.
8). a) (i) a recombinant nucleotide that encodes a Δ9 elongase polypeptide and has at least 80% amino acid identity based on the clustered V method of alignment when compared to the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 Providing a molecule, and (ii) a microbial host cell comprising a linoleic acid source;
b) expressing a nucleic acid fragment encoding a Δ9 elongase polypeptide and growing the microbial host cell of step (a) under conditions that convert linoleic acid to eicosadienoic acid;
c) optionally producing eicosadienoic acid comprising the step of recovering eicosadienoic acid of step (b).
9. a) (i) a recombinant nucleotide that encodes a Δ9 elongase polypeptide and has at least 80% amino acid identity based on the clustered V method of alignment when compared to the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14 Providing a molecule, and (ii) a microbial host cell comprising an alpha-linolenic acid source;
b) growing a microbial host cell of step (a) under conditions that express a nucleic acid fragment encoding a Δ9 elongase polypeptide and convert α-linolenic acid to eicosatrienoic acid;
c) optionally producing eicosatrienoic acid comprising step (b) recovering eicosatrienoic acid.
10. The microbial host cell is a Yarrowia species comprising a recombinant nucleotide molecule encoding a Δ9 elongase polypeptide set forth in SEQ ID NO: 26, wherein the recombinant nucleotide molecule is optimized for expression in Yarrowia The method of said 8 or 9 containing.
11. a. ) The recombinant nucleic acid molecule has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 26;
b. ) The host cell is Yarrowia lipolytica,
10. The method according to 8 or 9 above.
12 An isolated nucleic acid molecule which encodes the Δ9 elongase set forth in SEQ ID NO: 26 and wherein at least 98 codons are codon optimized for expression in Yarrowia species.

Claims (11)

Have a delta 9 elongase activity, and SEQ ID NO: 13 or SEQ ID NO: 1 4, a non-naturally occurring polynucleotide encoding a polypeptide comprising a least also an amino acid sequence having 90% identity based on the Clustal V method of alignment including, microbial host cell transformed. The microbial host cell of claim 1, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14. The microbial host cell of claim 2, wherein the non-natural polynucleotide comprises SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 26. The microbial host cell according to any one of claims 1 to 3, selected from the group consisting of yeast, algae, bacteria, Euglena, stramenopile, and fungi. The microbial host cell according to claim 4, wherein the cell is yeast. 6. The microbial host cell of claim 5, wherein the yeast is an oleaginous yeast selected from the group consisting of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichospolone, and Lipomyces. The microbial host cell according to claim 6, wherein the oleaginous yeast is Yarrowia lipolytica. a) A microbial host cell according to claim 1, and a step of further providing a microbial host cell comprising a re-Nord acid source,
b) expressing the polynucleotide that encoding a polypeptide having Δ9 Eronga peptidase activity comprising the steps of growing the microbial host cell of step (a) under conditions to convert linoleic acid to eicosadienoic acid,
A method for producing eicosadienoic acid , comprising: A microbial host cell according to any one of a) claims 1 to 7, and further alpha - and steps of providing a microbial host cell comprising a linolenic acid source,
b) expressing the polynucleotide that encoding a polypeptide having Δ9 Eronga peptidase activity, under conditions to convert the α- linolenic acid eicosatrienoic acid, comprising the steps of growing the microbial host cell of step (a) A process for producing eicosatrienoic acid , comprising: (I) the microbial host cell is a Yarrowia species, (ii) the polypeptide comprises SEQ ID NO: 27, and (iii) at least 98 polynucleotides optimized for expression in Yarrowia 10. A method according to claim 8 or 9, comprising a codon. The delta 9 elongase having an amino acid sequence set forth in SEQ ID NO 27 includes a code sul nucleotide sequence, even without less of the nucleotide sequence 98 codons are codon-optimized for expression in Yarrowia species An isolated nucleic acid molecule.

Images